U.S. patent application number 17/604708 was filed with the patent office on 2022-07-14 for novel radiolabelled cxcr4-targeting compounds for diagnosis and therapy.
The applicant listed for this patent is PROVINCIAL HEALTH SERVICES AUTHORITY, THE UNIVERSITY OF BRITISH COLUMBIA. Invention is credited to Francois BENARD, Daniel KWON, Joseph LAU, Kuo-Shyan LIN, Jerome LOZADA, Carlos Uribe MUNOZ, David PERRIN, Etienne ROUSSEAU, Zhengxing ZHANG.
Application Number | 20220218852 17/604708 |
Document ID | / |
Family ID | 1000006238359 |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220218852 |
Kind Code |
A1 |
BENARD; Francois ; et
al. |
July 14, 2022 |
NOVEL RADIOLABELLED CXCR4-TARGETING COMPOUNDS FOR DIAGNOSIS AND
THERAPY
Abstract
This application relates to compounds of Formula (I): [targeting
peptide]-N(R.sup.1)--X.sup.1(R.sup.2)L.sup.1-[linker]-R.sup.X.sub.n1
(I). The targeting peptide is
cyclo[L-Phe-L-Tyr-L-Lys(iPr)-D-Arg-L-2-Nal-Gly-D-Glu]-L-Lys(iPr).
R.sup.1 is H or methyl. X.sup.1 is an optionally substituted
C.sub.1-C.sub.15 hydrocarbon optionally comprising heteroatoms.
R.sup.2 is C(O)OH or C(O)NH.sub.2. L.sup.1 is a linkage
(thiolether, amide, maleimide-thiol, triazole). The linker has a
net negative charge at physiological pH and is a linear or branched
chain of 1-10 units of X.sup.2L.sup.2 and/or
X.sup.2(L.sup.2).sub.2, wherein: each X.sup.2 is, independently, an
optionally substituted C.sub.1-C.sub.15 hydrocarbon optionally
comprising heteroatoms; and each L.sup.2 is a linkage. The linker
optionally further comprises an albumin binder bonded to an
L.sup.2. Each R.sup.X is a radiolabelling group linked through a
separate L.sup.2, selected from: a metal chelator; a prosthetic
group containing trifluoroborate (BF.sub.3); or a prosthetic group
containing a silicon-fluorine-acceptor moiety. The compounds may be
useful for imaging CXCR4-expressing tissues or for treating
CXCR4-associated diseases or conditions (e.g. cancer).
Inventors: |
BENARD; Francois;
(Vancouver, CA) ; LIN; Kuo-Shyan; (Richmond,
CA) ; ROUSSEAU; Etienne; (Sherbrooke, CA) ;
ZHANG; Zhengxing; (Vancouver, CA) ; KWON; Daniel;
(Coquitlam, CA) ; LAU; Joseph; (Richmond, CA)
; MUNOZ; Carlos Uribe; (Surrey, CA) ; LOZADA;
Jerome; (Vancouver, CA) ; PERRIN; David;
(Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PROVINCIAL HEALTH SERVICES AUTHORITY
THE UNIVERSITY OF BRITISH COLUMBIA |
Vancouver
Vancouver |
|
CA
CA |
|
|
Family ID: |
1000006238359 |
Appl. No.: |
17/604708 |
Filed: |
April 17, 2020 |
PCT Filed: |
April 17, 2020 |
PCT NO: |
PCT/CA2020/050521 |
371 Date: |
October 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62835733 |
Apr 18, 2019 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 51/0482 20130101; A61K 51/088 20130101 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 51/04 20060101 A61K051/04; A61P 35/00 20060101
A61P035/00 |
Claims
1. A compound of Formula I or a salt or solvate of Formula I
[targeting
peptide]-N(R.sup.1)--X.sup.1(R.sup.2)L.sup.1-[linker]-R.sup.X.sub.n1
(I), wherein: the targeting peptide is
cyclo[L-Phe-L-Tyr-L-Lys(iPr)-D-Arg-L-2-NaI-Gly-D-Glu]-L-Lys(iPr)
which is C-terminally bonded to --N(R.sup.1)--; R.sup.1 is H or
methyl; X.sup.1 is a linear, branched, and/or cyclic
C.sub.1-C.sub.15 alkylenyl, alkenylenyl or alkynylenyl wherein 0-6
carbons are independently replaced by N, S, and/or O heteroatoms,
and substituted with 0-3 groups independently selected from one or
a combination of oxo, hydroxyl, sulfhydryl, halogen, guanidino,
carboxylic acid, sulfonic acid, sulfinic acid, and/or phosphoric
acid; R.sup.2 is C(O)OH or C(O)NH.sub.2; L.sup.1 is --S--,
--NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--,
##STR00181## the linker is a linear or branched chain of 1-10 units
of X.sup.2L.sup.2 and/or X.sup.2(L.sup.2).sub.2, wherein: each
X.sup.2 is, independently, a linear, branched, and/or cyclic
C.sub.1-C.sub.15 alkylenyl, alkenylenyl or alkynylenyl wherein 0-6
carbons are independently replaced by N, S, and/or O heteroatoms,
and substituted with 0-3 groups independently selected from one or
a combination of oxo, hydroxyl, sulfhydryl, halogen, guanidino,
carboxylic acid, sulfonic acid, sulfinic acid, and/or phosphoric
acid; each L.sup.2 is independently --S--, --NHC(O)--, --C(O)NH--,
--N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--, ##STR00182## the linker
comprises at least one carboxylic acid, sulfonic acid, sulfinic
acid, or phosphoric acid, and has a net negative charge at
physiological pH; the linker optionally further comprises an
albumin binder bonded to an L.sup.2 of the linker, wherein the
albumin binder is: --(CH.sub.2).sub.n2--CH.sub.3 wherein n2 is
8-20; --(CH.sub.2).sub.n3--C(O)OH wherein n3 is 8-20, or
##STR00183## wherein n4=1-4 and R.sup.3 is I, Br, F, Cl, H, OH,
OCH.sub.3, NH.sub.2, NO.sub.2 or CH.sub.3; n1 is 1 or 2; and each
R.sup.X is a radiolabelling group linked through a separate L.sub.2
of the linker, and is independently selected from: a metal chelator
optionally in complex with a radiometal or radioisotope-bound
metal; a prosthetic group containing trifluoroborate (BF.sub.3); or
a prosthetic group containing a silicon-fluorine-acceptor
moiety.
2. The compound of claim 1, wherein X.sup.1 is a linear, branched,
and/or cyclic C.sub.1-C.sub.15 alkylenyl.
3. The compound of claim 2, wherein X.sup.1 is ##STR00184##
4. The compound of claim 1, wherein
--N(R.sup.1)--X.sup.1(R.sup.2)L.sup.1- forms a sidechain-linked
amino acid residue selected from Lys, ornithine,
2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab),
Glu, Asp, or 2-aminoadipic acid (2-Aad).
5. The compound of any one of claims 1 to 4, wherein R.sup.1 is
H.
6. The compound of any one of claims 1 to 5, wherein R.sup.2 is
C(O)OH or C(O)NH.sub.2.
7. The compound of any one of claims 1 to 6, wherein L.sup.1 is
--NHC(O)-- or --C(O)NH--.
8. The compound of any one of claims 1 to 7, wherein the linker
consists of 1-8 units of X.sup.2L.sup.2 and 0-2 units of
X.sup.2(L.sup.2).sub.2.
9. The compound of any one of claims 1 to 8, wherein each X.sup.2
is independently a linear, branched, and/or cyclic C.sub.1-C.sub.15
alkylenyl.
10. The compound of any one of claims 1 to 7, wherein each X.sup.2
is independently: -CH--; ##STR00185## wherein each R.sup.4 is
independently carboxylic acid, sulfonic acid, sulfinic acid, or
phosphoric acid; or ##STR00186##
11. The compound of any one of claims 1 to 10, wherein each L.sup.2
between two X.sup.2 groups is independently --NHC(O)--, --C(O)NH--,
--N(CH.sub.3)C(O)--, or --C(O)N(CH.sub.3)--, and each L.sup.2
linking an R.sup.X is independently --S--, --NHC(O)--, --C(O)NH--,
--N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--, ##STR00187##
12. The compound of any one of claims 1 to 8, wherein the linker is
a linear or branched peptide of amino acid residues selected from
proteinogenic amino acid residues and/or nonproteinogenic amino
acid residues listed in Table 1, wherein each L.sup.2 between two
X.sup.2 groups is methylated or unmethylated, and wherein each
L.sup.2 linking an R.sup.X is independently --S--, --NHC(O)--,
--C(O)NH--, --N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--,
##STR00188##
13. The compound of claim 11 or 12, wherein each L.sup.2 between
two X.sup.2 groups is an unmethylated amide.
14. The compound of any one of claims 1 to 13, wherein the linker
comprises 2 or 3 amino acids selected from one or a combination of:
Glu, Asp, and/or 2-aminoadipic acid (2-Aad).
15. The compound of claim 14, wherein the linker comprises 3
consecutive Glu residues.
16. The compound of any one of claims 1 to 15, wherein the linker
has a net negative charge of -2 to -5 at physiological pH.
17. The compound of any one of claims 1 to 16, wherein the linker
further comprises the albumin binder.
18. The compound of any one of claims 1 to 17, wherein wherein each
L.sup.2 linking an R.sup.X is independently --NHC(O)--, --C(O)NH--,
##STR00189##
19. The compound of any one of claims 1 to 18, wherein n1 is 1.
20. The compound of any one of claims 1 to 18, wherein n1 is 2.
21. The compound of claim 20, comprising both the metal chelator
and the prosthetic group containing BF.sub.3.
22. The compound of claim 20, comprising two prosthetic groups each
containing a BF.sub.3.
23. The compound of any one of claims 1 to 22, wherein a prosthetic
group containing BF.sub.3 is --R.sup.6R.sup.7BF.sub.3 wherein
R.sup.6 is --(CH.sub.2).sub.1-5-- and --R.sup.7BF.sub.3 is selected
from Table 3 or 4 or is ##STR00190## wherein each R.sup.8 and each
R.sup.9 are independently a branched or linear C.sub.1-C.sub.5
alkyl.
24. The compound of claim 23, wherein --R.sup.7BF.sub.3 is
##STR00191##
25. The compound of claim 24, wherein R.sup.8 and R.sup.9 are each
methyl.
26. The compound of any one of claims 1 to 25, wherein the
prosthetic group containing BF.sub.3 comprises .sup.18F.
27. The compound of any one of claims 1 to 22, wherein the metal
chelator is in complex with the radioisotope.
28. The compound of any one of claims 1 to 22 or 27, wherein the
metal chelator is a polyaminocarboxylate chelator.
29. The compound of claim 28, wherein the metal chelator is DOTA or
a DOTA derivative.
30. The compound of claim 1, which has the structure of any one of
BL02, BL03, BL04, BL07, BL08, BL09, BL17, BL18, BL19, BL20, BL21,
BL22, BL23, BL24, BL25, BL26, BL27, BL28, or BL29, or which is a
salt or solvate thereof, wherein DOTA is optionally in complex with
a radioisotope or wherein the prosthetic group containing BF.sub.3
optionally comprises .sup.18F.
31. The compound of any one of claims 1 to 30, for use in imaging a
CXCR4-expressing tissue in a subject or for imaging an inflammatory
condition or disease, wherein at least one R.sup.X comprises or is
complexed with an imaging radioisotope.
32. The compound of any one of claims 1 to 30, for use in treating
a disease or condition characterized by expression of CXCR4 in a
subject, wherein at least one R.sup.X comprises or is complexed
with a therapeutic radioisotope.
33. The compound of claim 32, wherein the disease or condition is a
CXCR4-expressing cancer.
Description
FIELD OF INVENTION
[0001] The present invention relates to radiolabelled compounds for
selective imaging or treatment, particularly compounds that target
CXCR4.
BACKGROUND OF THE INVENTION
[0002] C-X-C chemokine receptor type 4 (CXCR4) is a G
protein-coupled receptor involved in chemotaxis and leukocyte
trafficking. CXCR4 was identified as a co-receptor for HIV entry
into T cells, establishing itself as a prominent target for
pharmaceutical development (Feng et al., Science. 1996, 272:872-7;
Bleul et al., Proc Natl Acad Sci. 1997, 94:1925-1930). The
expression of CXCR4 is also associated with autoimmune disorders,
cardiovascular disease and cancer (Doring et al., Front Physiol.
2014, 5:212; Chatterjee et al., Adv Cancer Res. 2014, 124:31-82),
including the observed overexpression of CXCR4 in 23 human cancers
including hematological and solid cancers (Chatterjee et al.,
ibid). The .alpha.-chemokine stromal cell-derived factor 1 (SDF-1a)
signals through CXCR4 to promote cancer cell proliferation and to
potentiate metastatic behavior (Duda et al., Clin Cancer Res. 2011,
17:2074-2080). Plerixafor, also known as AMD3100, developed
originally for HIV treatment, received FDA approval (De Clercq,
Biochem Pharmacol. 2009, 77:1655-1664) to mobilize hematopoietic
stem cells into peripheral blood for collection and autologous
transplantation.
[0003] Radiolabeled monoclonal antibodies, cyclam inhibitors, and
peptides have been used as pharmacophores for CXCR4-targeted
imaging in nuclear medicine (Weiss et al., Theranostics. 2013,
3:76-84; Walenkamp et al., J Nucl Med. 2017, 58:77S-82S). To date,
[.sup.68Ga]Ga-Pentixafor, a cyclic pentapeptide adapted by the
Wester group (Demmer et al., Chem Med Chem. 2011, 6:1789-1791;
Gourni et al., J Nucl Med. 2011, 52:1803-1810), is the most
investigated CXCR4 radiopharmaceutical in the clinic.
[.sup.68Ga]Ga-Pentixafor has been used to image patients with
leukemia, lymphoma, multiple myeloma, adrenocortical carcinoma,
small cell lung carcinoma, or breast carcinoma (Walenkamp et al.,
supra; Vag et al., EJNMMI Res. 2018, 8:90). Pentixather, a
derivative of Pentixafor with an iodinated tyrosine, is the
companion therapeutic agent (radiolabeled with .sup.177Lu-lutetium
or .sup.90Y-yttrium) for endoradiotherapy (Schottelius et al.,
Theranostics. 2017, 7:2350-2362; Herrmann et al., J Nucl Med. 2016,
57:248-251). Preliminary data with
[.sup.177Lu]Lu/[.sup.90Y]Y-Pentixather on a compassionate-use basis
was reported for three patients with refractory multiple myeloma
(Herrmann et al., ibid). Based on [.sup.18F]FDG imaging, one
patient had partial response and one had complete response. The
third patient failed to undergo [.sup.18F]FDG restaging due to
sepsis following autologous stem cell transplantation. Pending more
studies, [.sup.177Lu]Lu/[.sup.90Y]Y-Pentixather appears to be a
promising radiotherapeutic agent.
[0004] LY2510924
(cyclo[Phe-Tyr-Lys(iPr)-D-Arg-2-NaI-Gly-D-Glu]-Lys(iPr)-NH.sub.2)
is a novel cyclic peptide that can block SDF-1a binding to CXCR4
with an IC.sub.50 value of 79 pM (Peng et al., Mol Cancer Ther.
2015, 14:480-490). The authors demonstrated that LY2510924 was able
to inhibit growth of non-Hodgkin lymphoma, renal cell carcinoma,
lung cancer, colorectal cancer, and breast cancer xenograft models.
LY2510924 failed to improve treatment efficacy of
carboplatin/etoposide chemotherapy for small cell lung cancer
patients (Salgia et al., Lung Cancer. 2017, 105:7-13); however, it
is currently being evaluated in a phase II study in combination
with idarubicin and cytarabine for patients with relapsed or
refractory acute myeloid leukemia (ClinicalTrials.gov Identifier:
NCT02652871). In this regimen, LY2510924 is expected to mobilize
cancer cells from bone marrow to enter the bloodstream, where they
can be acted upon by the combination of chemotherapeutics.
[0005] There is therefore an unmet need in the field for improved
imaging agents (e.g. PET imaging agents) and radiotherapeutic
compositions for in-vivo diagnosis and treatment of cancers and
other diseases/disorders characterized by expression of CXCR4
[0006] No admission is necessarily intended, nor should it be
construed, that any of the preceding information constitutes prior
art against the present invention.
SUMMARY OF THE INVENTION
[0007] Disclosed herein are novel compounds targeting CXCR4.
[0008] This disclosure provides a compound, wherein the compound
has Formula I (shown below) or is a salt or a solvate of Formula
I
[targeting
peptide]-N(R.sup.1)--X.sup.1(R.sup.2)L.sup.1-[linker]-R.sup.X.sub.n1
(I),
wherein: the targeting peptide is
cyclo[L-Phe-L-Tyr-L-Lys(iPr)-D-Arg-L-2-NaI-Gly-D-Glu]-L-Lys(iPr)
which is C-terminally bonded to --N(R.sup.1)--; R.sup.1 is H or
methyl; X.sup.1 is a linear, branched, and/or cyclic
C.sub.1-C.sub.15 alkylenyl, alkenylenyl or alkynylenyl wherein 0-6
carbons are independently replaced by N, S, and/or O heteroatoms,
and substituted with 0-3 groups independently selected from one or
a combination of oxo, hydroxyl, sulfhydryl, halogen, guanidino,
carboxylic acid, sulfonic acid, sulfinic acid, and/or phosphoric
acid; R.sup.2 is C(O)OH or C(O)NH.sub.2; L.sup.1 is --S--,
--NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--,
--C(O)N(CH.sub.3)--,
##STR00001## [0009] the linker is a linear or branched chain of
1-10 units of X.sup.2L.sup.2 and/or X.sup.2(L.sup.2).sub.2,
wherein: each X.sup.2 is, independently, a linear, branched, and/or
cyclic C.sub.1-C.sub.15 alkylenyl, alkenylenyl or alkynylenyl
wherein 0-6 carbons are independently replaced by N, S, and/or O
heteroatoms, and substituted with 0-3 groups independently selected
from one or a combination of oxo, hydroxyl, sulfhydryl, halogen,
guanidino, carboxylic acid, sulfonic acid, sulfinic acid, and/or
phosphoric acid; [0010] each L.sup.2 is independently --S--,
--NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--,
--C(O)N(CH.sub.3)--,
[0010] ##STR00002## [0011] the linker comprises at least one
carboxylic acid, sulfonic acid, sulfinic acid, or phosphoric acid,
and has a net negative charge at physiological pH; [0012] the
linker optionally further comprises an albumin binder bonded to an
L.sup.2 of the linker, wherein the albumin binder is:
--(CH.sub.2).sub.n2--CH.sub.3 wherein n2 is 8-20;
--(CH.sub.2).sub.n3--C(O)OH wherein n3 is 8-20, or
##STR00003##
[0012] wherein n4=1-4 and R.sup.3 is I, Br, F, Cl, H, OH,
OCH.sub.3, NH.sub.2, NO.sub.2 or CH.sub.3; n1 is 1 or 2; and each
R.sup.X is a radiolabelling group linked through a separate L.sub.2
of the linker, and is independently selected from: a metal chelator
optionally in complex with a radiometal or radioisotope-bound
metal; a prosthetic group containing trifluoroborate (BF.sub.3); or
a prosthetic group containing a silicon-fluorine-acceptor
moiety.
[0013] This summary of the invention does not necessarily describe
all features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0015] FIG. 1 shows a graph of the percentage internalization of
the bound [.sup.68Ga]Ga-BL02 in CHO:CXCR4 and CHO:WT cells.
[0016] FIG. 2 shows maximal intensity projection PET images of
[.sup.68Ga]Ga-BL02 at A) 1 and B) 2 h post-injection in mice
bearing Daudi Burkitt's lymphoma xenografts. C) Blocking study was
performed by pre-injection of 7.5 .mu.g of LY2510924 (i.p.) 15
minutes before tracer administration. The scale bar is in units of
% ID/g from 0 to 6.
[0017] FIG. 3 shows maximal intensity projection PET images of
[.sup.68Ga]Ga-BL02 at A) 1 h post-injection in mice bearing Z138
mantle cell lymphoma xenografts. B) Blocking study was performed by
pre-injection of 7.5 .mu.g of LY2510924 (i.p.) 15 minutes before
tracer administration. The scale bar is in units of % ID/g from 0
to 11.
[0018] FIG. 4 shows maximal intensity projection PET images of
[.sup.68Ga]Ga-BL02 at A) 1 h post-injection in mice bearing Jeko1
mantle cell lymphoma xenografts. B) Blocking study was performed by
pre-injection of 7.5 .mu.g of LY2510924 (i.p.) 15 minutes before
tracer administration. The scale bar is in units of % ID/g from 0
to 11.
[0019] FIG. 5 shows maximal intensity projection PET images of
[.sup.68Ga]Ga-BL02 at A) 1 h post-injection in mice bearing
GRANTA519 mantle cell lymphoma xenografts. B) Blocking study was
performed by pre-injection of 7.5 .mu.g of LY2510924 (i.p.) 15
minutes before tracer administration. The scale bar is in units of
% ID/g from 0 to 5.
[0020] FIG. 6 shows maximal intensity projection PET images of
[.sup.68Ga]Ga-BL02 at A) 1 h post-injection in mice bearing PC3
prostate adenocarcinoma xenografts. B) Blocking study was performed
by pre-injection of 7.5 .mu.g of LY2510924 (i.p.) 15 minutes before
tracer administration. The scale bar is in units of % ID/g from 0
to 1.5.
[0021] FIG. 7 shows maximal intensity projection PET images of
[.sup.18F]F-BL04 at A) 1 and B) 2 h post-injection in mice bearing
Daudi Burkitt's lymphoma xenografts. C) Blocking study was
performed by pre-injection of 7.5 .mu.g of LY2510924 15 min (i.p.)
before tracer administration. The scale bar is in units of % ID/g
from 0 to 5.
[0022] FIG. 8 shows maximal intensity projection PET images of
[.sup.68Ga]Ga-BL06 at A) 1 and B) 2 h post-injection in mice
bearing Daudi Burkitt's lymphoma xenografts. C) Blocking study was
performed by pre-injection of 7.5 .mu.g of LY2510924 15 min (i.p.)
before tracer administration. The scale bar is in units of % ID/g
from 0 to 10.
[0023] FIG. 9 shows maximal intensity projection PET images of
[.sup.18F]F-BL08 at A) 1 and B) 2 h post-injection in mice bearing
Daudi Burkitt's lymphoma xenografts. C) Blocking study was
performed by pre-injection of 7.5 .mu.g of LY2510924 15 min (i.p.)
before tracer administration. The scale bar is in units of % ID/g
from 0 to 9.
[0024] FIG. 10 shows maximal intensity projection PET images of
[.sup.18F]F-BL09 at A) 1 and B) 2 h post-injection in mice bearing
Daudi Burkitt's lymphoma xenografts. C) Blocking study was
performed by pre-injection of 7.5 .mu.g of LY2510924 15 min (i.p.)
before tracer administration. The scale bar is in units of % ID/g
from 0 to 9.
[0025] FIG. 11 shows a maximal intensity projection PET image of
[.sup.68Ga]Ga-BL17 at 1 h post-injection in mice bearing Daudi
Burkitt's lymphoma xenografts. The scale bar is in units of % ID/g
from 0 to 6.
DETAILED DESCRIPTION
[0026] As used herein, the terms "comprising," "having",
"including" and "containing," and grammatical variations thereof,
are inclusive or open-ended and do not exclude additional,
unrecited elements and/or method steps. The term "consisting
essentially of" if used herein in connection with a composition,
use or method, denotes that additional elements and/or method steps
may be present, but that these additions do not materially affect
the manner in which the recited composition, method or use
functions. The term "consisting of" if used herein in connection
with a composition, use or method, excludes the presence of
additional elements and/or method steps. A composition, use or
method described herein as comprising certain elements and/or steps
may also, in certain embodiments consist essentially of those
elements and/or steps, and in other embodiments consist of those
elements and/or steps, whether or not these embodiments are
specifically referred to. A use or method described herein as
comprising certain elements and/or steps may also, in certain
embodiments consist essentially of those elements and/or steps, and
in other embodiments consist of those elements and/or steps,
whether or not these embodiments are specifically referred to.
[0027] A reference to an element by the indefinite article "a" does
not exclude the possibility that more than one of the elements is
present, unless the context clearly requires that there be one and
only one of the elements. The singular forms "a", "an", and "the"
include plural referents unless the content clearly dictates
otherwise. The use of the word "a" or "an" when used herein in
conjunction with the term "comprising" may mean "one," but it is
also consistent with the meaning of "one or more," "at least one"
and "one or more than one."
[0028] Unless otherwise specified, "certain embodiments", "various
embodiments", "an embodiment" and similar terms includes the
particular feature(s) described for that embodiment either alone or
in combination with any other embodiment or embodiments described
herein, whether or not the other embodiments are directly or
indirectly referenced and regardless of whether the feature or
embodiment is described in the context of a method, product, use,
composition, compound, et cetera.
[0029] As used herein, the terms "treat", "treatment",
"therapeutic" and the like includes ameliorating symptoms, reducing
disease progression, improving prognosis and reducing recurrence
(e.g. reducing cancer recurrence).
[0030] As used herein, the term "diagnostic agent" includes an
"imaging agent". As such, a "diagnostic radiometal" includes
radiometals that are suitable for use in imaging agents and
"diagnostic radioisotope" includes radioisotopes that are suitable
for use in imaging agents.
[0031] The term "subject" refers to an animal (e.g. a mammal or a
non-mammal animal). The subject may be a human or a non-human
primate. The subject may be a laboratory mammal (e.g., mouse, rat,
rabbit, hamster and the like). The subject may be an agricultural
animal (e.g., equine, ovine, bovine, porcine, camelid and the like)
or a domestic animal (e.g., canine, feline and the like). In some
embodiments, the subject is a human.
[0032] The compounds disclosed herein may also include base-free
forms, salts or pharmaceutically acceptable salts thereof. Unless
otherwise specified, the compounds claimed and described herein are
meant to include all racemic mixtures and all individual
enantiomers or combinations thereof, whether or not they are
explicitly represented herein.
[0033] The compounds disclosed herein may be shown as having one or
more charged groups, may be shown with ionizable groups in an
uncharged (e.g. protonated) state or may be shown without
specifying formal charges. As will be appreciated by the person of
skill in the art, the ionization state of certain groups within a
compound (e.g. without limitation, carboxylic acid, sulfonic acid,
sulfinic acid, phosphoric acid and the like) is dependent, inter
alia, on the pKa of that group and the pH at that location. For
example, but without limitation, a carboxylic acid group (i.e.
COOH) would be understood to usually be deprotonated (and
negatively charged) at neutral pH and at most physiological pH
values, unless the protonated state is stabilized. Likewise,
sulfonic acid groups, sulfinic acid groups, and phosphoric acid
groups would generally be deprotonated (and negatively charged) at
neutral and physiological pH values.
[0034] As used herein, the terms "salt" and "solvate" have their
usual meaning in chemistry. As such, when the compound is a salt or
solvate, it is associated with a suitable counter-ion. It is well
known in the art how to prepare salts or to exchange counter-ions.
Generally, such salts can be prepared by reacting free acid forms
of these compounds with a stoichiometric amount of a suitable base
(e.g. without limitation, Na, Ca, Mg, or K hydroxide, carbonate,
bicarbonate, or the like), or by reacting free base forms of these
compounds with a stoichiometric amount of a suitable acid. Such
reactions are generally carried out in water or in an organic
solvent, or in a mixture of the two. Counter-ions may be changed,
for example, by ion-exchange techniques such as ion-exchange
chromatography. All zwitterions, salts, solvates and counter-ions
are intended, unless a particular form is specifically
indicated.
[0035] In certain embodiments, the salt or counter-ion may be
pharmaceutically acceptable, for administration to a subject. More
generally, with respect to any pharmaceutical composition disclosed
herein, non-limiting examples of suitable excipients include any
suitable buffers, stabilizing agents, salts, antioxidants,
complexing agents, tonicity agents, cryoprotectants,
lyoprotectants, suspending agents, emulsifying agents,
antimicrobial agents, preservatives, chelating agents, binding
agents, surfactants, wetting agents, non-aqueous vehicles such as
fixed oils, or polymers for sustained or controlled release. See,
for example, Berge et al. 1977. (J. Pharm Sci. 66:1-19), or
Remington--The Science and Practice of Pharmacy, 21st edition
(Gennaro et al editors. Lippincott Williams & Wilkins
Philadelphia), each of which is incorporated by reference in its
entirety.
[0036] As used herein, the expression "Cy-Cz", where y and z are
integers (e.g. C.sub.1-C.sub.15, C.sub.1-C.sub.5, and the like),
refers to the number of carbons in a compound, R-group or
substituent, or refers to the number of carbons plus heteroatoms
when a certain number of carbons are specified as being replaced by
heteroatoms. Heteroatoms may include any, some or all possible
heteroatoms. For example, in some embodiments, the heteroatoms are
selected from N, O, S, P and Se. In some embodiments, the
heteroatoms are selected from N, S and O. Unless otherwise
specified, such embodiments are non-limiting.
[0037] Unless explicitly stated otherwise, the term "alkyl"
includes any reasonable combination of the following: (1) linear or
branched; (2) acyclic or cyclic, the latter of which may include
multi-cyclic (fused rings, multiple non-fused rings or a
combination thereof; and (3) unsubstituted or substituted. In the
context of the expression "alkyl, alkenyl or alkynyl" and similar
expressions, the "alkyl" would be understood to be a saturated
alkyl. As used herein, the term "linear" may be used as it is
normally understood to a person of skill in the art and generally
refers to a chemical entity that comprises a skeleton or main chain
that does not split off into more than one contiguous chain.
Non-limiting examples of linear alkyls include methyl, ethyl,
n-propyl, and n-butyl. As used herein, the term "branched" may be
used as it is normally understood to a person of skill in the art
and generally refers to a chemical entity that comprises a skeleton
or main chain that splits off into more than one contiguous chain.
The portions of the skeleton or main chain that split off in more
than one direction may be linear, cyclic or any combination
thereof. Non-limiting examples of a branched alkyl group include
tert-butyl and isopropyl.
[0038] The term "alkylenyl" refers to a divalent analog of an alkyl
group. In the context of the expression "alkylenyl, alkenylenyl or
alkynylenyl", and similar expressions, the "alkylenyl" would be
understood to be a saturated alkylenyl.
[0039] As used herein, the term "saturated" when referring to a
chemical entity may be used as it is normally understood to a
person of skill in the art and generally refers to a chemical
entity that comprises only single bonds, and may include linear,
branched, and/or cyclic groups. Non-limiting examples of a
saturated C.sub.1-C.sub.20 alkyl group may include methyl, ethyl,
n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl,
t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl,
i-hexyl, 1,2-dimethylpropyl, 2-ethylpropyl, 1-methyl-2-ethylpropyl,
I-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl,
1,1,2-triethylpropyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl,
2-ethylbutyl, 1,3-dimethylbutyl, 2-methylpentyl, 3-methylpentyl,
sec-hexyl, t-hexyl, n-heptyl, i-heptyl, sec-heptyl, t-heptyl,
n-octyl, i-octyl, sec-octyl, t-octyl, n-nonyl, i-nonyl, sec-nonyl,
t-nonyl, n-decyl, i-decyl, sec-decyl, t-decyl, cyclopropanyl,
cyclobutanyl, cyclopentanyl, cyclohexanyl, cycloheptanyl,
cyclooctanyl, cyclononanyl, cyclodecanyl, and the like. Unless
otherwise specified, a C.sub.1-C.sub.20 alkylenyl therefore
encompasses, without limitation, all divalent analogs of the
above-listed saturated alkyl groups.
[0040] As used herein, the term "unsaturated" when referring to a
chemical entity may be used as it is normally understood to a
person of skill in the art and generally refers to a chemical
entity that comprises at least one double or triple bond, and may
include linear, branched, and/or cyclic groups. Non-limiting
examples of a C.sub.2-C.sub.20 alkenyl group may include vinyl,
allyl, isopropenyl, I-propene-2-yl, 1-butene-1-yl, 1-butene-2-yl,
1-butene-3-yl, 2-butene-1-yl, 2-butene-2-yl, octenyl, decenyl,
cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl,
cycloheptenyl, cyclooctenyl, cyclononanenyl, cyclodecanenyl, and
the like. Unless otherwise specified, a C.sub.1-C.sub.20
alkenylenyl therefore encompasses, without limitation, all divalent
analogs of the above-listed alkenyl groups. Non-limiting examples
of a C.sub.2-C.sub.20 alkynyl group may include ethynyl, propynyl,
butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl,
and the like. Unless otherwise specified, a C.sub.1-C.sub.20
alkynylenyl therefore encompasses, without limitation, all divalent
analogs of the above-listed alkynyl groups.
[0041] Where it is specified that 1 or more carbons in an alkyl,
alkenyl, alkynyl, alkylenyl, alkenylenyl, alkynylenyl, etc., are
independently replaced by a heteroatom, the person of skill in the
art would understand that various combinations of different
heteroatoms may be used. Non-limiting examples of non-aromatic
heterocyclic groups include aziridinyl, azetidinyl, diazetidinyl,
pyrrolidinyl, pyrrolinyl, piperidinyl, piperazinyl, imidazolinyl,
pyrazolidinyl, imidazolydinyl, phthalimidyl, succinimidyl,
oxiranyl, tetrahydropyranyl, oxetanyl, dioxanyl, thietanyl,
thiepinyl, morpholinyl, oxathiolanyl, and the like. The expression
"a linear, branched, and/or cyclic . . . alkyl, alkenyl or alkynyl"
includes, inter alia, aryl groups. Unless further specified, an
"aryl" group includes both single aromatic rings as well as fused
rings containing at least one aromatic ring. non-limiting examples
of C.sub.3-C.sub.20 aryl groups include phenyl (Ph), pentalenyl,
indenyl, naphthyl and azulenyl. Non-limiting examples of
X.sub.3-X.sub.20 aromatic heterocyclic groups include pyrrolyl,
imidazolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl,
pirazinyl, quinolinyl, isoquinolinyl, acridinyl, indolyl,
isoindolyl, indolizinyl, purinyl, carbazolyl, indazolyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, phenanthridinyl, phenazinyl,
phenanthrolinyl, perimidinyl, furyl, dibenzofuryl, xanthenyl,
benzofuryl, thiophenyl, thianthrenyl, benzothiophenyl,
phosphorinyl, phosphinolinyl, phosphindolyl, thiazolyl, oxazolyl,
isoxazolyl, and the like. Likewise, the expression "a linear,
branched, and/or cyclic . . . alkylenyl, alkenylenyl or
alkynylenyl" includes, interalia, divalent analogs of the
above-defined linear, branched, and/or cyclic alkyl, alkenyl or
alkynyl groups, including all aryl groups encompassed therein.
[0042] As used herein, the term "substituted" is used as it would
normally be understood to a person of skill in the art and
generally refers to a compound or chemical entity that has one
chemical group replaced with a different chemical group. Unless
otherwise specified, a substituted alkyl is an alkyl in which one
or more hydrogen atom(s) are independently each replaced with an
atom that is not hydrogen. For example, chloromethyl is a
non-limiting example of a substituted alkyl, more particularly an
example of a substituted methyl. Aminoethyl is another non-limiting
example of a substituted alkyl, more particularly an example of a
substituted ethyl. Unless otherwise specified, a substituted
compound or group (e.g. alkyl, alkylenyl, aryl, and the like) may
be substituted with any chemical group reasonable to the person of
skill in the art. For example, but without limitation, a hydrogen
bonded to a carbon or heteroatom (e.g. N) may be substituted with
halide (e.g. F, I, Br, Cl), amine, amide, oxo, hydroxyl, thiol
(sulfhydryl), phosphate (or phosphoric acid), phosphonate, sulfate,
SO.sub.2H (sulfinic acid), SO.sub.3H (sulfonic acid), alkyls, aryl,
ketones, carboxaldehyde, carboxylic acid, carboxamides, nitriles,
guanidino, monohalomethyl, dihalomethyl or trihalomethyl.
[0043] As used herein, the term "unsubstituted" is used as it would
normally be understood to a person of skill in the art.
Non-limiting examples of unsubstituted alkyls include methyl,
ethyl, tert-butyl, pentyl and the like. The expression "optionally
substituted" is used interchangeably with the expression
"unsubstituted or substituted".
[0044] In the structures provided herein, hydrogen may or may not
be shown. In some embodiments, hydrogens (whether shown or
implicit) may be protium (i.e. .sup.1H), deuterium (i.e. .sup.2H)
or combinations of .sup.1H and .sup.2H. Methods for exchanging
.sup.1H with .sup.2H are well known in the art. For
solvent-exchangeable hydrogens, the exchange of .sup.1H with
.sup.2H occurs readily in the presence of a suitable deuterium
source, without any catalyst. The use of acid, base or metal
catalysts, coupled with conditions of increased temperature and
pressure, can facilitate the exchange of non-exchangeable hydrogen
atoms, generally resulting in the exchange of all .sup.1H to
.sup.2H in a molecule.
[0045] The compounds disclosed herein incorporate amino acids, e.g.
as residues in a peptide chain (linear or branched) or as amino
acids that are otherwise part of a compound. Amino acids have both
an amino group and a carboxylic acid group, either or both of which
can be used for covalent attachment. In attaching to the remainder
of the compound, the amino group and/or the carboxylic acid group
may be converted to an amide or other structure; e.g. a carboxylic
acid group of a first amino acid is converted to an amide (e.g. a
peptide bond) when bonded to the amino group of a second amino
acid. As such, amino acid residues may have the formula
--N(R.sup.a)R.sup.bC(O)--, where R.sup.a and R.sup.b are R-groups.
R.sup.a will typically be hydrogen or methyl. The amino acid
residues of a peptide may comprise typical peptide (amide) bonds
and may further comprise bonds between side chain functional groups
and the side chain or main chain functional group of another amino
acid. For example, the side chain carboxylate of one amino acid
residue in the peptide (e.g. Asp, Glu, etc.) may be bonded to and
the amine of another amino acid residue in the peptide (e.g. Dap,
Dab, Orn, Lys). Further details are provided below. The term "amino
acid" includes proteinogenic and nonproteinogenic amino acids.
Non-limiting examples of nonproteinogenic amino acids are shown in
Table 1 and include: D-amino acids (including without limitation
any D-form of the following amino acids), ornithine (Orn),
3-(1-naphtyl)alanine (NaI), 3-(2-naphtyl)alanine (2-NaI),
.alpha.-aminobutyric acid, norvaline, norleucine (Nle),
homonorleucine, beta-(1,2,3-triazol-4-yl)-L-alanine,
1,2,4-triazole-3-alanine, Phe(4-F), Phe(4-CI), Phe(4-Br), Phe(4-1),
Phe(4-NH.sub.2), Phe(4-NO.sub.2), homoarginine (hArg),
2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic
acid (Agp), B-alanine, 4-aminobutyric acid, 5-aminovaleric acid,
6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid,
9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid,
2-amino-3-(anthracen-2-yl)propanoic acid,
2-amino-3-(anthracen-9-yl)propanoic acid,
2-amino-3-(pyren-1-yl)propanoic acid, Trp(5-Br), Trp(5-OCH.sub.3),
Trp(6-F), Trp(5-OH) or Trp(CHO), 2-aminoadipic acid (2-Aad),
3-aminoadipic acid (3-Aad), propargylglycine (Pra),
homopropargylglycine (Hpg), beta-homopropargylglycine (Bpg),
2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab),
azidolysine (Lys(N.sub.3)), azido-ornithine (Orn(N.sub.3)),
2-amino-4-azidobutanoic acid Dab(N.sub.3), Dap(N.sub.3),
2-(5'-azidopentyl)alanine, 2-(6'-azidohexyl)alanine,
4-amino-1-carboxymethyl-piperidine (Pip),
4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp), and tranexamic
acid. If not specified as an L- or D-amino acid, an amino acid
shall be understood to encompass both L- and D-amino acids.
TABLE-US-00001 TABLE 1 List of non-limiting examples of
non-proteinogenic amino acids. Any D-amino acid of a proteinogenic
amino acid 10-aminodecanoic acid ornithine (Orn) 2-aminooctanoic
acid 3-(1-naphtyl)alanine (Nal) 2-amino-3-(anthracen-2-yl)propanoic
acid 3-(2-naphtyl)alanine (2-Nal)
2-amino-3-(anthracen-9-yl)propanoic acid .alpha.-aminobutyric acid
2-amino-3-(pyren-1-yl)propanoic acid norvaline Trp(5-Br),
norleucine (Nle) Trp(5-OCH.sub.3), homonorleucine Trp(6-F),
beta-(1,2,3-triazol-4-yl)-L-alanine Trp(5-OH)
1,2,4-triazole-3-alanine Trp(CHO), Phe(4-F), 2-aminoadipic acid
(2-Aad) Phe(4-Cl), 3-aminoadipic acid (3-Aad) Phe(4-Br),
propargylglycine (Pra) Phe(4-I), homopropargylglycine (Hpg)
Phe(4-NH.sub.2), beta-homopropargylglycine (Bpg) Phe(4-NO.sub.2),
2,3-diaminopropionic acid (Dap) homoarginine (hArg)
2,4-diaminobutyric acid (Dab)
4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp) azidolysine
(Lys(N.sub.3)) 2-(5'-azidopentyl)alanine azido-ornithine
(Orn(N.sub.3)) 2-(6'-azidohexyl)alanine amino-4-azidobutanoic acid
Dab(N.sub.3) 2-amino-4-guanidinobutyric acid (Agb) tranexamic acid
2-amino-3-guanidinopropionic acid (Agp)
4-amino-1-carboxymethyl-piperidine (Pip) .beta.-alanine
NH.sub.2(CH.sub.2).sub.2O(CH.sub.2).sub.2C(O)OH 4-aminobutyric acid
NH.sub.2(CH.sub.2).sub.2[O(CH.sub.2).sub.2].sub.2C(O)OH
5-aminovaleric acid
NH.sub.2(CH.sub.2).sub.2[O(CH.sub.2).sub.2].sub.3C(O)OH
6-aminohexanoic acid
NH.sub.2(CH.sub.2).sub.2[O(CH.sub.2).sub.2].sub.4C(O)OH
7-aminoheptanoic acid
NH.sub.2(CH.sub.2).sub.2[O(CH.sub.2).sub.2].sub.5C(O)OH
8-aminooctanoic acid
NH.sub.2(CH.sub.2).sub.2[O(CH.sub.2).sub.2].sub.6C(O)OH
9-aminononanoic acid
[0046] The wavy line "" symbol shown through or at the end of a
bond in a chemical formula (e.g. in the definitions L.sup.1,
L.sup.2, etc.) is intended to define the R group on one side of the
wavy line, without modifying the definition of the structure on the
opposite side of the wavy line. Where an R group is bonded on two
or more sides (e.g. certain definitions of X.sup.1, X.sup.2, etc.),
any atoms shown outside the wavy lines are intended to clarify
orientation of the R group. As such, only the atoms between the two
wavy lines constitute the definition of the R group. When atoms are
not shown outside the wavy lines, or for a chemical group shown
without wavy lines but does have bonds on multiple sides (e.g.
--C(O)NH--, and the like.), the chemical group should be read from
left to right matching the orientation in the formula that the
group relates to (e.g. for formula --R.sup.a-R.sup.b-R.sup.c--, the
definition of R.sup.b as --C(O)NH-- would be incorporated into the
formula as --R.sup.a--C(O)NH--R.sup.c-- not as
--R.sup.a--NHC(O)--R.sup.c--).
[0047] In various aspects, there is disclosed a compound, wherein
the compound has Formula I or is a salt or a solvate of Formula
I:
[targeting
peptide]-N(R.sup.1)--X.sup.1(R.sup.2)L.sup.1-[linker]-R.sup.X.sub.n1
(1),
wherein: the targeting peptide is
cyclo[L-Phe-L-Tyr-L-Lys(iPr)-D-Arg-L-2-NaI-Gly-D-Glu]-L-Lys(iPr)
which is C-terminally bonded to --N(R.sup.1)--; R.sup.1 is H or
methyl; X.sup.1 is a linear, branched, and/or cyclic
C.sub.1-C.sub.15 hydrocarbon (e.g. alkylenyl, alkenylenyl or
alkynylenyl) wherein 0-6 carbons are independently replaced by N,
S, and/or O heteroatoms, and substituted with 0-3 groups
independently selected from one or a combination of oxo, hydroxyl,
sulfhydryl, halogen, guanidino, carboxylic acid, sulfonic acid,
sulfinic acid, and/or phosphoric acid; R.sup.2 is C(O)OH or
C(O)NH.sub.2; L.sup.1 is --S--, --NHC(O)--, --C(O)NH--,
--N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--,
##STR00004##
the linker is a linear or branched chain of 1-10 units of
X.sup.2L.sup.2 and/or X.sup.2(L.sup.2).sub.2, wherein: [0048] each
X.sup.2 is, independently, a linear, branched, and/or cyclic
C.sub.1-C.sub.15 hydrocarbon (e.g. alkylenyl, alkenylenyl or
alkynylenyl) wherein 0-6 carbons are independently replaced by N,
S, and/or O heteroatoms, and substituted with 0-3 groups
independently selected from one or a combination of oxo, hydroxyl,
sulfhydryl, halogen, guanidino, carboxylic acid, sulfonic acid,
sulfinic acid, and/or phosphoric acid; [0049] each L.sup.2 is
independently --S--, --NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--,
--C(O)N(CH.sub.3)--,
[0049] ##STR00005## [0050] the linker comprises at least one
carboxylic acid, sulfonic acid, sulfinic acid, or phosphoric acid,
and has a net negative charge at physiological pH; [0051] the
linker optionally further comprises an albumin binder bonded to an
L.sub.2 of the linker, wherein the albumin binder is:
--(CH.sub.2).sub.n2--CH.sub.3 wherein n2 is 8-20;
--(CH.sub.2).sub.n3--C(O)OH wherein n3 is 8-20, or
##STR00006##
[0051] wherein n4=1-4 and R.sup.3 is I, Br, F, Cl, H, OH,
OCH.sub.3, NH.sub.2, NO.sub.2 or CH.sub.3; n1 is 1 or 2; and each
R.sup.X is a radiolabelling group linked through a separate L.sub.2
of the linker, and is independently selected from: a metal chelator
optionally in complex with a radiometal or radioisotope-bound
metal; a prosthetic group containing trifluoroborate (BF.sub.3); or
a prosthetic group containing a silicon-fluorine-acceptor
moiety.
[0052] The targeting peptide has the structure of Formula II or is
a salt or solvate of Formula II:
##STR00007##
[0053] In some embodiments, R.sup.1 is H. In other embodiments,
R.sup.1 is methyl.
[0054] X.sup.1 is a linear, branched, and/or cyclic
C.sub.1-C.sub.15 hydrocarbon (e.g. alkylenyl, alkenylenyl or
alkynylenyl) wherein 0-6 carbons are independently replaced by N,
S, and/or O heteroatoms, and substituted with 0-3 groups
independently selected from one or a combination of oxo, hydroxyl,
sulfhydryl, halogen, guanidino, carboxylic acid, sulfonic acid,
sulfinic acid, and/or phosphoric acid. In some embodiments, the
hydrocarbon is an alkylenyl. In some embodiments, the hydrocarbon
is an alkenylenyl. In some embodiments, the hydrocarbon is an
alkynylenyl. In some embodiments, the hydrocarbon is linear. In
some embodiments, the hydrocarbon is branched. In some embodiments,
the hydrocarbon is cyclic. The term "cyclic" in this context
includes single ring, multi-ring or fused ring systems, each of
which can individually be aromatic, partially aromatic or
non-aromatic. In some embodiments, the hydrocarbon is linear and
cyclic. In some embodiments, the hydrocarbon is branched and
cyclic.
[0055] In some embodiments, X.sup.1 is a linear, branched, and/or
cyclic C.sub.1-C.sub.15 alkylenyl. In some embodiments, X.sup.1 is
a linear alkylenyl. In some embodiments, X.sup.1 is
##STR00008##
In some embodiments, X.sup.1 is
##STR00009##
In some embodiments, X.sup.1 is
##STR00010##
[0056] In some embodiments, --N(R.sup.1)--X.sup.1(R.sup.2)L.sup.1-
forms a sidechain-linked amino acid residue. In some embodiments,
the sidechain-linked amino acid residue is Lys, ornithine,
2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab),
Glu, Asp, or 2-aminoadipic acid (2-Aad). In some embodiments, the
sidechain-linked amino acid residue is an L-amino acid. In some
embodiments, the sidechain-linked amino acid residue is a D-amino
acid. In some embodiments, the sidechain-linked amino acid residue
is L-Lys. In some embodiments, the sidechain-linked amino acid
residue is D-Lys.
[0057] In some embodiments, R.sup.2 is C(O)OH. In other
embodiments, R.sup.2 is C(O)NH.sub.2.
[0058] L.sup.1 is a linkage selected from --S--, --NHC(O)--,
--C(O)NH--, --N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--,
##STR00011##
In some embodiments, L.sup.1 is --S--. In some embodiments, L.sup.1
is --NHC(O)--. In some embodiments, L.sup.1 is --C(O)NH--. In some
embodiments, L.sup.1 is --N(CH.sub.3)C(O)--. In some embodiments,
L.sup.1 is --C(O)N(CH.sub.3)--. In some embodiments, L.sup.1 is
##STR00012##
In some embodiments, L.sup.1 is
##STR00013##
In some embodiments, L.sup.1 is
##STR00014##
In some embodiments, L.sup.1 is
##STR00015##
[0059] The "linker" is a linear or branched chain of 1-10 units of
X.sup.2L.sup.2 and/or X.sup.2(L.sup.2).sub.2, including any
combination or configuration of X.sup.2L.sup.2 and/or
X.sup.2(L.sup.2).sub.2. In some embodiments, the linker consists
only of X.sup.2L.sup.2 units (e.g. 1-10 units of X.sup.2L.sup.2 and
zero units of X.sup.2(L.sup.2).sub.2). In some embodiments, the
linker has 3 units of X.sup.2L.sup.2. In some embodiments, the
linker has 1 unit of X.sup.2(L.sup.2).sub.2. In some embodiments,
the linker has 2 units of X.sup.2(L.sup.2).sub.2. In some
embodiments, the linker has 3 units of X.sup.2(L.sup.2).sub.2. In
some embodiments, the linker has 1-8 units of X.sup.2L.sup.2 and
0-2 units of X.sup.2(L.sup.2).sub.2. In some embodiments, the
linker has 1-3 units of X.sup.2L.sup.2 and 0 units of
X.sup.2(L.sup.2).sub.2. In some embodiments, the linker has 3 units
of X.sup.2L.sup.2 and 0 units of X.sup.2(L.sup.2).sub.2. In some
embodiments, the linker has 4 units of X.sup.2L.sup.2 and 0 units
of X.sup.2(L.sup.2).sub.2. In some embodiments, the linker has 1
units of X.sup.2L.sup.2 and 1 unit of X.sup.2(L.sup.2).sub.2. In
some embodiments, the linker has 2 units of X.sup.2L.sup.2 and 1
unit of X.sup.2(L.sup.2).sub.2. In some embodiments, the linker has
3 units of X.sup.2L.sup.2 and 1 unit of X.sup.2(L.sup.2).sub.2. In
some embodiments, the linker has 4 units of X.sup.2L.sup.2 and 1
unit of X.sup.2(L.sup.2).sub.2. In some embodiments, the linker has
5 units of X.sup.2L.sup.2 and 1 unit of X.sup.2(L.sup.2).sub.2. In
some embodiments, the linker has 6 units of X.sup.2L.sup.2 and 1
unit of X.sup.2(L.sup.2).sub.2. In some embodiments, the linker has
7 units of X.sup.2L.sup.2 and 1 unit of X.sup.2(L.sup.2).sub.2. In
some embodiments, the linker has 1-8 units of X.sup.2L.sup.2 and 2
units of X.sup.2(L.sup.2).sub.2.
[0060] Each X.sup.2 is, independently, a linear, branched, and/or
cyclic C.sub.1-C.sub.15 hydrocarbon (e.g. alkylenyl, alkenylenyl or
alkynylenyl) wherein 0-6 carbons are independently replaced by N,
S, and/or O heteroatoms, and substituted with 0-3 groups
independently selected from one or a combination of oxo, hydroxyl,
sulfhydryl, halogen, guanidino, carboxylic acid, sulfonic acid,
sulfinic acid, and/or phosphoric acid. In some embodiments, one or
more hydrocarbon is an alkylenyl. In some embodiments, one or more
hydrocarbon is an alkenylenyl. In some embodiments, one or more
hydrocarbon is an alkynylenyl. In some embodiments, one or more
hydrocarbon is linear and cyclic. In some embodiments, one or more
hydrocarbon is branched and cyclic. The term "cyclic" in this
context includes single ring, multi-ring or fused ring systems,
each of which can individually be aromatic, partially aromatic or
non-aromatic. In some embodiments, each hydrocarbon is linear.
[0061] In some embodiments, each X.sup.2 in each X.sup.2L.sup.2
unit is independently a linear, branched, and/or cyclic
C.sub.1-C.sub.15 alkylenyl. In some embodiments, each X.sup.2 in
each X.sup.2L.sup.2 unit is, independently, a linear or branched
C.sub.1-C.sub.15 alkylenyl substituted with 0-1 group independently
selected from carboxylic acid, sulfonic acid, sulfinic acid, and/or
phosphoric acid. In some embodiments, each X.sup.2 in each
X.sup.2L.sup.2 unit is, independently, a linear or branched
C.sub.2-C.sub.6 alkylenyl substituted with 0-1 group independently
selected from carboxylic acid, sulfonic acid, sulfinic acid, and/or
phosphoric acid. In some embodiments, each X.sup.2 in each
X.sup.2L.sup.2 unit is, independently, a linear or branched
C.sub.2-C.sub.6 alkylenyl substituted with 0-1 carboxylic acid
group.
[0062] In some embodiments, each X.sup.2 in each X.sup.2L.sup.2
unit is independently a linear, branched, and/or cyclic
C.sub.1-C.sub.15 alkylenyl. In some embodiments, each X.sup.2 in
each X.sup.2(L.sup.2).sub.2 unit is, independently, a linear or
branched C.sub.1-C.sub.15 alkylenyl. In some embodiments, each
X.sup.2 in each X.sup.2(L.sup.2).sub.2 unit is, independently, a
linear or branched C.sub.2-C.sub.6 alkylenyl.
[0063] In some embodiments, each X.sup.2 is independently:
--CH(R)-- wherein each R is independently H or C.sub.1-C.sub.3
linear or branched alkyl;
##STR00016##
wherein each R.sup.4 is independently hydrogen, carboxylic acid,
sulfonic acid, sulfinic acid, or phosphoric acid; or
##STR00017##
In some embodiments, each X.sup.2 is independently: --CH--;
##STR00018##
wherein each R.sup.4 is independently carboxylic acid, sulfonic
acid, sulfinic acid, or phosphoric acid; or
##STR00019##
In some embodiments, each X.sup.2 is independently: --CH--;
##STR00020##
[0064] Each L.sup.2 is a linkage independently selected from --S--,
--NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--,
--C(O)N(CH.sub.3)--,
##STR00021##
In some embodiments, each L.sup.2 between two X.sup.2 groups is
independently --NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--, or
--C(O)N(CH.sub.3)--, and each L.sup.2 linking R.sup.X is
independently --S--, --NHC(O)--, --C(O)NH--,
##STR00022##
In some such embodiments, each L.sup.2 linking R.sup.X is
independently-NHC(O)--, --C(O)NH--, --N(CH.sub.3)C(O)--,
--C(O)N(CH.sub.3)--,
##STR00023##
In some such embodiments, each L.sup.2 linking R.sup.X is
independently-NHC(O)--, --C(O)NH--,
##STR00024##
In some such embodiments, each L.sup.2 between two X.sup.2 groups
is an unmethylated amide. In some such embodiments, 1, 2, 3, 4, or
5 instances of L.sup.2 between two X.sup.2 groups is a methylated
amide.
[0065] In some embodiments, the linker (when including the --C(O)--
of L.sup.1) corresponds to a linear or branched peptide of amino
acid residues selected from proteinogenic amino acid residues
and/or nonproteinogenic amino acid residues (e.g. as listed in
Table 1), and wherein each L.sup.2 between two X.sup.2 groups is
methylated or unmethylated, and wherein each L.sup.2 linking an
R.sup.X is independently --S--, --NHC(O)--, --C(O)NH--,
--N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--,
##STR00025##
In some such embodiments, each L.sup.2 between two X.sup.2 groups
is an unmethylated amide. In some such embodiments, 1, 2, 3, 4, or
5 instances of L.sup.2 between two X.sup.2 groups is a methylated
amide.
[0066] The amino acid residues in the linker may be all L-amino
acids, all D-amino acids, or a combination of L- and D-amino acids.
In some embodiments, all amino acids in the linker are L-amino
acids. In some embodiments, all amino acids in the linker are
D-amino acids.
[0067] In some embodiments, the linker comprises 2-7 amino acid
residues selected from one or a combination of: Glu, Asp, and/or
2-aminoadipic acid (2-Aad). In some embodiments, the linker
comprises 2 amino acid residues selected from one or a combination
of: Glu, Asp, and/or 2-Aad. In some embodiments, the linker
comprises 3 amino acid residues selected from one or a combination
of: Glu, Asp, and/or 2-Aad. In some embodiments, the linker
comprises 4 amino acid residues selected from one or a combination
of: Glu, Asp, and/or 2-Aad. In some embodiments, the linker
comprises 5 amino acid residues selected from one or a combination
of: Glu, Asp, and/or 2-Aad. In some embodiments, the linker
comprises 2 or 3 consecutive Glu, Asp, and/or 2-Aad residues. In
some embodiments, the linker comprises 3 consecutive Glu residues.
In some embodiments, the linker (when including the --C(O)-- of
L.sup.1) consists of a linear peptide of 3 Glu/Asp/2-Aad residues
(see compounds BL02, BL08, BL09, BL17, BL20, BL25).
[0068] In some embodiments, the linker has a net negative charge of
-1 to -5 at physiological pH. In some embodiments, the linker has a
net negative charge of -2 to -5 at physiological pH. In some
embodiments, the linker has a net negative charge of -1 at
physiological pH. In some embodiments, the linker has a net
negative charge of -2 at physiological pH. In some embodiments, the
linker has a net negative charge of -3 at physiological pH. In some
embodiments, the linker has a net negative charge of -4 at
physiological pH. In some embodiments, the linker has a net
negative charge of -5 at physiological pH.
[0069] In some embodiments, the linker has the structure of the
linker of any one of BL02, BL03, BL04, BL07, BL08, BL09, BL17,
BL18, BL19, BL20, BL21, BL22, BL23, BL24, BL25, BL26, BL27, BL28,
or BL29, or wherein the linker is a salt or solvate of a linker of
the foregoing.
[0070] In some embodiments, the compound has the structure of any
one of BL02, BL03, BL04, BL07, BL08, BL09, BL17, BL18, BL19, BL20,
BL21, BL22, BL23, BL24, BL25, BL26, BL27, BL28, or BL29, or which
is a salt or solvate thereof, wherein DOTA is optionally in complex
with a radioisotope or wherein the prosthetic group containing
BF.sub.3 optionally comprises .sup.18F.
[0071] In some embodiments, the linker further comprises an albumin
binder bonded to an L.sup.2 of the linker. In some embodiments, the
albumin binder is: --(CH.sub.2).sub.n2--CH.sub.3 wherein n2 is
8-20. In some embodiments, n2 is 12-18. In some embodiments, n2 is
14-18. In some embodiments, n2 is 16. In some embodiments, the
albumin binder is --(CH.sub.2).sub.n3--C(O)OH wherein n3 is 8-20.
In some embodiments, n3 is 12-18. In some embodiments, n3 is 14-18.
In some embodiments, n3 is 16. In some embodiments, the albumin
binder is
##STR00026##
wherein n4=1-4 and R.sup.3 is I, Br, F, Cl, H, OH, OCH.sub.3,
NH.sub.2, NO.sub.2 or CH.sub.3. In some embodiments, n4 is 1. In
some embodiments, n4 is 2. In some embodiments, n4 is 3. In some
embodiments, n4 is 4. In some embodiments, R.sup.3 is H, I, Cl, F,
OCH.sub.3, or CH.sub.3. In some embodiments, n4 is 3 and R.sup.3 is
H, I, Cl, F, OCH.sub.3, or CH.sub.3. In some embodiments, the
L.sup.2 incorporating the albumin binder into the linker is an
amide.
[0072] In some embodiments, n1 is 1. In other embodiments, n1 is 2;
i.e. the compound has two radiolabeling groups attached to the
linker. In some embodiments, the two radiolabeling groups are
different. In some embodiments, the two radiolabeling groups are
the same.
[0073] In some embodiments, an R.sup.X comprises a metal chelator
optionally in complex with a radiometal (e.g. .sup.68Ga or
.sup.177Lu) or in complex with a radioisotope-bound metal (e.g.
Al.sup.18F). The chelator may be any metal chelator suitable for
binding to the radiometal or to the metal-containing prosthetic
group bonded to the radioisotope (e.g. polyaminocarboxylates and
the like). Many suitable chelators are known, e.g. as summarized in
Price and Orvig, Chem. Soc. Rev., 2014, 43, 260-290, which is
incorporated by reference in its entirety. Non-limiting examples of
suitable chelators include those selected from the group consisting
of: DOTA and derivatives; DOTAGA; NOTA; NODAGA; NODASA; CB-DO2A;
3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogues optionally selected
from CHX-A''-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO;
CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; sarcophagine and sarcophagine
derivatives optionally selected from SarAr, SarAr-NCS, diamSar,
AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives;
H2-macropa or a derivative thereof; H.sub.2dedpa, H.sub.4octapa,
H.sub.4py4pa, H.sub.4Pypa, H.sub.2azapa, Hsdecapa, and other
picolinic acid derivatives; CP256; PCTA; C-NETA; C-NE3TA; HBED;
SHBED; BCPA; CP256; YM103; desferrioxamine (DFO) and DFO
derivatives; and H.sub.6phospa. Exemplary non-limiting examples of
suitable chelators and example radioisotopes (radiometals) chelated
by these chelators are shown in Table 2. In alternative
embodiments, an R.sup.X comprises a chelator selected from those
listed above or in Table 2, or is any other suitable chelator. One
skilled in the art could replace any of the chelators listed herein
with another chelator.
TABLE-US-00002 TABLE 2 Exemplary chelators and exemplary isotopes
which bind said chelators. Chelator Isotopes ##STR00027## Cu-64/67
Ga-67/68 In-111 Lu-177 Y-86/90 Bi-203/212/213 Pb-212 Ac-225 Gd-159
Yb-175 Ho-166 As-211 Sc-44/47 Pm-149 Pr-142 Sn-117m Sm-153
Tb-149/161 Er-165 Ra-223/224 Th-227 ##STR00028## Cu-64/67
##STR00029## Pb-212 ##STR00030## Bi-212/213 ##STR00031## Cu-64/67
##STR00032## Cu-64/67 ##STR00033## Cu-64/67 ##STR00034## Cu-64/67
##STR00035## Cu-64/67 Ga-68 In-111 Sc-44/47 ##STR00036## Cu-64/67
Ga-68 Lu-177 Y-86/90 Bi-213 Pb-212 ##STR00037## Au-198/199
##STR00038## Rh-105 ##STR00039## In-111 Sc-44/47 Lu-177 Y-86/90
Sn-117m Pd-109 ##STR00040## In-111 Lu-177 Y-86/90 Bi-212/213
##STR00041## Cu-64/67 ##STR00042## Cu-64/67 ##STR00043## In-111
Lu-177 Y-86/90 Ac-225 ##STR00044## Ac-225 ##STR00045## In-111
Ac-225 ##STR00046## In-111 Lu-177 Ac-225 ##STR00047## In-111 Lu-177
Ac-225 ##STR00048## In-111 Ga-68 ##STR00049## In-111 ##STR00050##
Cu-64/67 H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridil)methyl]- Ac-225
4,13-diaza-18-crown-6) ##STR00051##
[0074] In some embodiments, an R.sup.X of the compound is a
polyaminocarboxylate chelator. In some such embodiments, the
chelator is attached through an amide bond. In some embodiments,
R.sup.X is: DOTA or a derivative thereof; TETA or a derivative
thereof; SarAr or a derivative thereof; NOTA or a derivative
thereof; TRAP or a derivative thereof; HBED or a derivative
thereof; 2,3-HOPO or a derivative thereof; PCTA
(3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-tria-
cetic acid) or a derivative thereof; DFO or a derivative thereof;
DTPA or a derivative thereof; OCTAPA
(N,N0-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N0-diacetic
acid) or a derivative thereof; or H2-MACROPA or a derivative
thereof. In some embodiments, an R.sup.X is DOTA. In some
embodiments, an R.sup.X is a chelator moiety in complex with
radioisotope X wherein X is .sup.64Cu, .sup.67Cu, .sup.90Y,
.sup.111In, .sup.114mIn .sup.117mSn, .sup.153Sm, .sup.149Tb,
.sup.161Tb, .sup.177Lu .sup.225Ac, .sup.213Bi, .sup.224Ra,
.sup.212Bi, .sup.212Pb, .sup.227Th, .sup.223Ra, .sup.47Sc,
.sup.186Re or .sup.188Re. In some embodiments, X is .sup.177Lu. In
some embodiments, an R.sup.X is a chelator moiety in complex with
radioisotope X wherein X is .sup.64Cu, .sup.68Ga, .sup.86Y,
.sup.111In, .sup.94mTc, .sup.44Sc, .sup.89Zr, or .sup.99mTc. In
some embodiments, X is .sup.68Ga.
[0075] In some embodiments, the chelator is conjugated with a
radioisotope. The conjugated radioisotope may be, without
limitation, .sup.68Ga, .sup.61Cu, .sup.64Cu, .sup.67Ga, .sup.99mTc,
.sup.111In, .sup.44Sc, .sup.86Y, .sup.89Zr, .sup.90Nb, .sup.177Lu,
.sup.117mSn, .sup.165Er, .sup.90Y, .sup.227Th, .sup.225Ac,
.sup.213Bi, .sup.212Bi, .sup.211As, .sup.203Pb, .sup.212Pb,
.sup.47Sc, .sup.166Ho, .sup.188Re, .sup.186Re, .sup.149Pm,
.sup.159Gd, .sup.105Rh, .sup.109Pd, .sup.198Au, .sup.199Au,
.sup.175Yb, .sup.142Pr, .sup.14mIn, and the like. In some
embodiments, the chelator is a chelator from Table 2 and the
conjugated radioisotope is a radioisotope indicated in Table 2 as a
binder of the chelator.
[0076] In some embodiments, the chelator is not conjugated to a
radioisotope.
[0077] In some embodiments, the chelator is: DOTA or a derivative
thereof, conjugated with .sup.177Lu, .sup.111In, .sup.213Bi,
.sup.68Ga, .sup.67Ga, .sup.203Pb, .sup.212Pb, .sup.44Sc, .sup.47Sc,
.sup.90Y, .sup.86Y, .sup.225Ac, .sup.117mSn, .sup.153Sm,
.sup.149Tb, .sup.161Tb, .sup.165Er, .sup.224Ra, .sup.212Bi,
.sup.227Th, .sup.223Ra, .sup.64Cu or .sup.67Cu; H2-MACROPA
conjugated with .sup.225Ac; Me-3,2-HOPO conjugated with .sup.227Th;
H.sub.4py4pa conjugated with .sup.225Ac, .sup.227Th or .sup.177Lu;
H.sub.4pypa conjugated with .sup.177Lu; NODAGA conjugated with
.sup.68Ga; DTPA conjugated with .sup.111In; or DFO conjugated with
.sup.89Zr.
[0078] In some embodiments, the chelator is TETA, SarAr, NOTA,
TRAP, HBED, 2,3-HOPO, PCTA, DFO, DTPA, OCTAPA or another picolinic
acid derivative.
[0079] In some embodiments, an R.sup.X is a chelator for
radiolabelling with .sup.99mTc, .sup.94mTc, .sup.186Re, or
.sup.188Re, such as mercaptoacetyl, hydrazinonicotinamide,
dimercaptosuccinic acid, 1,2-ethylenediylbis-L-cysteine diethyl
ester, methylenediphosphonate, hexamethylpropyleneamineoxime and
hexakis(methoxy isobutyl isonitrile), and the like. In some
embodiments, an R.sup.X is a chelator, wherein the chelator is
mercaptoacetyl, hydrazinonicotinamide, dimercaptosuccinic acid,
1,2-ethylenediylbis-L-cysteine diethyl ester,
methylenediphosphonate, hexamethylpropyleneamineoxime or
hexakis(methoxy isobutyl isonitrile). In some of these embodiments,
the chelator is bound by a radioisotope. In some such embodiments,
the radioisotope is .sup.99mTc, .sup.94mTc, .sup.186Re, or
.sup.188Re.
[0080] In some embodiments, an R.sup.X is a chelator that can bind
.sup.18F-aluminum fluoride ([.sup.18F]AIF), such as
1,4,7-triazacyclononane-1,4-diacetate (NODA) and the like. In some
embodiments, the chelator is NODA. In some embodiments, the
chelator is bound by [.sup.18F]AIF.
[0081] In some embodiments, an R.sup.X is a chelator that can bind
.sup.72As or .sup.77As, such as a trithiol chelate and the like. In
some embodiments, the chelator is a trithiol chelate. In some
embodiments, the chelator is conjugated to .sup.72As. In some
embodiments, the chelator is conjugated to .sup.77As.
[0082] In some embodiments, an R.sup.X is a prosthetic group
containing a trifluoroborate (BF.sub.3), capable of
.sup.18F/.sup.19F exchange radiolabeling. Such an R.sup.X group may
be the only R.sup.X (n1=1), or may be in addition to second R.sup.X
(n1=2), wherein the second R.sup.X is the same or different as the
first R.sup.X. The prosthetic group may be R.sup.6R.sup.7BF.sub.3,
wherein R.sup.6 is independently --(CH.sub.2).sub.1-5-- and the
group --R.sup.7BF.sub.3 may independently be selected from one or a
combination of those listed in Table 3 (below), Table 4 (below),
or
##STR00052##
wherein R.sup.8 and R.sup.9 are independently C.sub.1-C.sub.5
linear or branched alkyl groups. For Tables 3 and 4, the R in the
pyridine substituted with --OR, --SR, --NR--, --NHR or --NR.sub.2
groups is C.sub.1-C.sub.5 branched or linear alkyl. In some
embodiments, --R.sup.7BF.sub.3 is selected from those listed in
Table 3. In some embodiments, --R.sup.7BF.sub.3 is independently
selected from one or a combination of those listed in Table 4. In
some embodiments, one fluorine is .sup.18F. In some embodiments,
all three fluorines are .sup.19F.
TABLE-US-00003 TABLE 3 Exemplary R.sup.7BF.sub.3 groups.
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087##
##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## ##STR00094## ##STR00095## ##STR00096##
TABLE-US-00004 TABLE 4 Exemplary R.sup.7BF.sub.3 groups.
##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101##
##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106##
##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111##
##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116##
##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121##
##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126##
##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131##
##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136##
##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141##
##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146##
##STR00147## ##STR00148##
[0083] In some embodiments, R.sup.7BF.sub.3 may form
##STR00149## ##STR00150## ##STR00151##
in which the R (when present) in the pyridine substituted --OR,
--SR, --NR--, --NHR or --NR.sub.2 is a branched or linear
C.sub.1-C.sub.5 alkyl. In some embodiments, R is a branched or
linear C.sub.1-C.sub.5 saturated alkyl. In some embodiments, R is
methyl. In some embodiments, R is ethyl. In some embodiments, R is
propyl. In some embodiments, R is isopropyl. In some embodiments, R
is n-butyl. In some embodiments, one fluorine is .sup.18F. In some
embodiments, all three fluorines are .sup.19F.
[0084] In some embodiments, R.sup.7BF.sub.3 may form
##STR00152## ##STR00153## ##STR00154##
in which the R (when present) in the pyridine substituted --OR,
--SR, --NR-- or --NR.sub.2 is branched or linear C.sub.1-C.sub.5
alkyl. In some embodiments, R is a branched or linear
C.sub.1-C.sub.5 saturated alkyl. In some embodiments, R is methyl.
In some embodiments, R is ethyl. In some embodiments, R is propyl.
In some embodiments, R is isopropyl. In some embodiments, R is
n-butyl. In some embodiments, --R.sup.7BF.sub.3 is
##STR00155##
In some embodiments, one fluorine is .sup.18F. In some embodiments,
all three fluorines are .sup.19F.
[0085] In some embodiments, --R.sup.7BF.sub.3 is
##STR00156##
In some embodiments, R.sup.8 is methyl. In some embodiments,
R.sup.8 is ethyl. In some embodiments, R.sup.8 is propyl. In some
embodiments, R.sup.8 is isopropyl. In some embodiments, R.sup.8 is
butyl. In some embodiments, R.sup.8 is n-butyl. In some
embodiments, R.sup.8 is pentyl. In some embodiments, R.sup.9 is
methyl. In some embodiments, R.sup.9 is ethyl. In some embodiments,
R.sup.9 is propyl. In some embodiments, R.sup.9 is isopropyl. In
some embodiments, R.sup.9 is butyl. In some embodiments, R.sup.9 is
n-butyl. In some embodiments, R.sup.9 is pentyl. In some
embodiments, R.sup.8 and R.sup.9 are both methyl. In some
embodiments, one fluorine is .sup.18F. In some embodiments, all
three fluorines are .sup.19F.
[0086] In some embodiments, an R.sup.X is a prosthetic group
containing a silicon-fluorine-acceptor moiety. In some embodiments,
the fluorine of the silicon-fluorine acceptor moiety is .sup.18F.
The prosthetic groups containing a silicon-fluorine-acceptor moiety
may be independently selected from one or a combination of the
following:
##STR00157##
wherein R.sup.11 and R.sup.12 are independently a linear or
branched, cyclic or acyclic, and/or aromatic or non-aromatic
C.sub.1-C.sub.10 alkyl, alkenyl or alkynyl group. In some
embodiments, R.sup.11 and R.sup.12 are independently selected from
the group consisting of phenyl, tert-butyl, sec-propyl or methyl.
In some embodiments, the prosthetic group is
##STR00158##
In some embodiments, the prosthetic group is
##STR00159##
In some embodiments, the prosthetic group is
##STR00160##
In some embodiments, the prosthetic group is
##STR00161##
[0087] The overexpression of CXCR4 has been observed in over 23
types of malignancies, including brain, breast, and prostate
cancers. Moreover, leukemia, lymphoma and myeloma have significant
CXCR4 expression. Retrospective studies have shown that CXCR4
expression is correlated with lowered survival for prostate and
melanoma patients. Furthermore, CXCR4 expression is a prognostic
factor of disease relapse for acute and chronic myeloid leukemia,
acute myelogenous leukemia and multiple myeloma. The SDF-1/CXCR4
axis mediates cancer growth, potentiates metastasis, recruits
stromal and immune cells to support malignant growth, and confers
chemotherapeutic resistance. Radiolabeled CXCR4 probes could be
used in the early diagnosis of solid and hematological malignancies
that express CXCR4. Such imaging agents could be used to confirm
the diagnostic of malignancy, or guide focal ablative treatment if
the disease is localized. Such ligands could also be used to
monitor response to therapy, by providing an independent assessment
of the residual cellular content of a tumour known to overexpress
CXCR4. [.sup.68Ga]Ga-Pentixafor has been used by the Wester group
for cancer imaging and to identify potential responders to
endoradiotherapy.
[0088] Dysregulation of the SDF-1/CXCR4 axis also mediates a number
of inflammatory conditions. In rheumatoid arthritis (RA),
SDF-1/CXCR4 signaling is responsible for the pro-inflammatory
migration of activated T-cells into the site of inflammation;
specifically, the synovium of patients with RA showed that the
presence of T-cells with increased expression of CXCR4. Given the
burden of RA on the population with respect to morbidity and
mortality, there is a significant amount of research into
developing therapeutics to mediate the inflammatory response,
especially with novel biologics being approved by the FDA in the
past few years. Radiolabeled CXCR4 probes for positron emission
tomography imaging would enable diagnosis and prognosis of the
rheumatoid arthritis and also be used to monitor therapy of
emerging disease-modifying antirheumatic drugs in clinical trials.
CXCR4 expression has been detected with PET imaging using
[r.sup.8Ga]Ga-Pentixafor in diseases with an inflammatory
component, including infectious bone diseases, urinary tract
infections as a complication after kidney transplantation,
myocardial infarctions, and ischemic strokes. CXCR4 imaging may
have a significant role in diagnosing and monitoring other
inflammatory diseases in the future.
[0089] In the setting of cardiac pathology, inflammatory diseases
of the cardiac vessel walls are mediated in part by the
dysregulation of the SDF-1/CXCR4 axis. In the early stages of
atherosclerosis, the SDF-1/CXCR4 axis recruits endothelial
progenitor cells towards sites of peripheral vascular damage,
thereby initiating plaque formation, though there is some evidence
towards an atheroprotective effect. Atherosclerotic plaques are
characterized by the presence of hypoxia, which has been shown to
upregulate the expression of CXCR4 and influence cell trafficking.
Finally, in a rabbit model of atherosclerosis,
[.sup.68Ga]Ga-Pentixafor enabled visualization of atherosclerotic
plaques by PET. In the same study, atherosclerotic plaques were
identified in patients with a history of atherosclerosis using
[.sup.68Ga]Ga-Pentixafor. As such, PET diagnostic agents targeting
CXCR4 are potentially viable as an alternative method of diagnosing
and obtaining prognostic information about atherosclerosis.
[0090] In certain embodiments, the compound is conjugated with a
radioisotope for positron emission tomography (PET) or single
photon emission computed tomography (SPECT) imaging of a
CXCR4-expressing tissue or for imaging an inflammatory condition or
disease (e.g. rheumatoid arthritis or cardiovascular disease),
wherein the compound is conjugated with a radioisotope that is a
positron emitter or a gamma emitter. Without limitation, the
positron or gamma emitting radioisotope may be .sup.68Ga,
.sup.67Ga, .sup.61Cu, .sup.64Cu, .sup.99mTc, .sup.110mIn,
.sup.111In, .sup.44Sc, .sup.86Y .sup.89Zr, .sup.90Nb, .sup.18F,
.sup.131I, .sup.123I, .sup.124I or .sup.72As.
[0091] When the radioisotope (e.g. X) is a diagnostic radioisotope,
there is disclosed use of certain embodiments of the compound for
preparation of a radiolabelled tracer for imaging. There is also
disclosed a method of imaging CXCR4-expressing tissues or an
inflammatory condition or disease in a subject, in which the method
comprises: administering to the subject a composition comprising
certain embodiments of the compound and a pharmaceutically
acceptable excipient; and imaging the subject, e.g. using positron
emission tomography (PET). When the tissue is a diseased tissue
(e.g. a CXCR4-expressing cancer), CXCR4-targeted treatment may then
be selected for treating the subject. There is therefore disclosed
the use of certain compounds of the invention in imaging a
CXCR4-expressing cancer in a subject, wherein R.sup.X comprises or
is complexed with a diagnostic or imaging radioisotope. In some
embodiments, the subject is human.
[0092] Given the broad expression of CXCR4 in cancers, there has
been a significant push to develop CXCR4-targeting therapeutics.
While CXCR4 inhibitors have demonstrated efficacy in tumor models
in mice, in both treating tumors and preventing metastasis, very
few pharmaceutical agents have demonstrated efficacy in clinical
trials. Plerixafor, also known as AMD3100, developed originally for
HIV treatment, is the lone CXCR4 antagonist to receive FDA approval
to date. AMD3100 is given to lymphoma and multiple myeloma patients
to mobilize hematopoietic stem cells into peripheral blood for
collection and autologous transplantation, and not as a method of
direct treatment. There is an unmet clinical need for treating
CXCR4-expressing cancers, many of which are resistant to the
standard of care available today.
[0093] Cancers that are CXCR4 positive could be susceptible to
endoradiotherapy. In this application, a peptide targeting CXCR4 is
radiolabeled with a radioisotope, usually a .beta.- or
.alpha.-particle emitter, to deliver a high local dose of radiation
to lesions. These radioactive emissions usually inflict DNA damage,
thereby inducing cellular death. This method of therapy has been
exploited in oncology, with the somatostatin receptor (for
neuroendocrine tumors) and prostate-specific membrane antigen (for
metastatic castration-resistant prostate cancer) being two
examples. Unlike external beam radiation therapy, this systemic
treatment can be effective even in the metastatic setting.
Therapeutic radioisotopes include but are not restricted to
.sup.177Lu, .sup.90Y, .sup.225Ac and .sup.64Cu.
[0094] With respect to cardiac pathologies, a small retrospective
study with endoradiotherapy by [.sup.90Y]Y- or
[.sup.177Lu]Lu-Pentixather demonstrated regression of CXCR4
expression and activity in patients with previously identified
atherosclerotic plaques. Therefore, radionuclide therapy may
present a novel route of therapy for inflammatory diseases such as
atherosclerosis.
[0095] In certain embodiments the compound is conjugated with a
radioisotope that is used for therapy (e.g. cancer therapy). This
includes radioisotopes such as .sup.165Er, .sup.212Bi, .sup.211At,
.sup.166Ho, .sup.149Pm, .sup.159Gd, .sup.105Rh, .sup.109Pd
.sup.198Au, .sup.199Au, .sup.175Yb, .sup.142Pr, .sup.177Lu
(.beta.-emitter, t.sub.2/1=6.65 d), .sup.111In, .sup.213Bi,
.sup.203Pb, .sup.212Pb, .sup.47Sc, .sup.90Y (1-emitter,
t.sub.2/1=2.66 d), .sup.117mSn, .sup.153Sm, .sup.149Tb, .sup.161Tb,
.sup.224Ra, .sup.225Ac (a-emitter, t.sub.2/1=9.95 d), .sup.227Th,
.sup.223Ra, .sup.77As, .sup.131I, .sup.64Cu or .sup.67Cu.
[0096] When the radioisotope (e.g. X) is a therapeutic
radioisotope, there is disclosed use of certain embodiments of the
compound (or a pharmaceutical composition thereof) for the
treatment of a disease or condition characterized by expression of
CXCR4 in a subject. Accordingly, there is provided use of the
compound in preparation of a medicament for treating a disease or
condition characterized by expression of CXCR4 in a subject. There
is also provided a method of treating a disease or condition
characterized by expression of CXCR4 in a subject, in which the
method comprises: administering to the subject a composition
comprising the compound and a pharmaceutically acceptable
excipient. For example, but without limitation, the disease may be
a CXCR4-expressing cancer (e.g. non-Hodgkin lymphoma, lymphoma,
multiple myeloma, leukemia, adrenocortical cancer, lung cancer,
breast cancer, renal cell cancer, colorectal cancer). There is
therefore disclosed the use of certain compounds of the invention
for treating a CXCR4-expressing cancer in a subject, wherein
R.sup.Xcomprises or is complexed with a therapeutic radioisotope.
In some embodiments, the subject is human.
[0097] The compounds presented herein incorporate peptides, which
may be synthesized by any of a variety of methods established in
the art. This includes but is not limited to liquid-phase as well
as solid-phase peptide synthesis using methods employing
9-fluorenylmethoxycarbonyl (Fmoc) and/or t-butyloxycarbonyl (Boc)
chemistries, and/or other synthetic approaches.
[0098] Solid-phase peptide synthesis methods and technology are
well-established in the art. For example, peptides may be
synthesized by sequential incorporation of the amino acid residues
of interest one at a time. In such methods, peptide synthesis is
typically initiated by attaching the C-terminal amino acid of the
peptide of interest to a suitable resin. Prior to this, reactive
side chain and alpha amino groups of the amino acids are protected
from reaction by suitable protecting groups, allowing only the
alpha carboxyl group to react with a functional group such as an
amine group, a hydroxyl group, or an alkyl halide group on the
solid support. Following coupling of the C-terminal amino acid to
the support, the protecting group on the side chain and/or the
alpha amino group of the amino acid is selectively removed,
allowing the coupling of the next amino acid of interest. This
process is repeated until the desired peptide is fully synthesized,
at which point the peptide can be deprotected and cleaved from the
support, and purified. A non-limiting example of an instrument for
solid-phase peptide synthesis is the Aapptec Endeavor 90 peptide
synthesizer.
[0099] To allow coupling of additional amino acids, Fmoc protecting
groups may be removed from the amino acid on the solid support,
e.g. under mild basic conditions, such as piperidine (20-50% v/v)
in DMF. The amino acid to be added must also have been activated
for coupling (e.g. at the alpha carboxylate). Non-limiting examples
of activating reagents include without limitation
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU),
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU),
benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate
(BOP),
benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosp-
hate (PyBOP). Racemization is minimized by using triazoles, such as
1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole
(HOAt). Coupling may be performed in the presence of a suitable
base, such as N,N-diisopropylethylamine (DIPEA/DIEA) and the like.
For long peptides or if desired, peptide synthesis and ligation may
be used.
[0100] Apart from forming typical peptide bonds to elongate a
peptide, peptides may be elongated in a branched fashion by
attaching to side chain functional groups (e.g. carboxylic acid
groups or amino groups), either: side chain to side chain; or side
chain to backbone amino or carboxylate. Coupling to amino acid side
chains may be performed by any known method, and may be performed
on-resin or off-resin. Non-limiting examples include: forming an
amide between an amino acid side chain containing a carboxyl group
(e.g. Asp, D-Asp, Glu, D-Glu, Aad, and the like) and an amino acid
side chain containing an amino group (e.g. Lys, D-Lys, Orn, D-Orn,
Dab, D-Dab, Dap, D-Dap, and the like) or the peptide N-terminus;
forming an amide between an amino acid side chain containing an
amino group (e.g. Lys, D-Lys, Orn, D-Orn, Dab, D-Dab, Dap, D-Dap,
and the like) and either an amino acid side chain containing a
carboxyl group (e.g. Asp, D-Asp, Glu, D-Glu, and the like) or the
peptide C-terminus; and forming a 1, 2, 3-triazole via click
chemistry between an amino acid side chain containing an azide
group (e.g. Lys(N.sub.3), D-Lys(N.sub.3), and the like) and an
alkyne group (e.g. Pra, D-Pra, and the like). The protecting groups
on the appropriate functional groups must be selectively removed
before amide bond formation, whereas the reaction between an alkyne
and an azido groups via the click reaction to form an
1,2,3-triazole does not require selective deprotection.
Non-limiting examples of selectively removable protecting groups
include 2-phenylisopropyl esters (O-2-PhiPr) (e.g. on Asp/Glu) as
well as 4-methyltrityl (Mtt), allyloxycarbonyl (alloc),
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene))ethyl (Dde), and
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)
(e.g. on Lys/Orn/Dab/Dap). O-2-PhiPr and Mtt protecting groups can
be selectively deprotected under mild acidic conditions, such as
2.5% trifluoroacetic acid (TFA) in DCM. Alloc protecting groups can
be selectively deprotected using
tetrakis(triphenylphosphine)palladium(0) and phenylsilane in DCM.
Dde and ivDde protecting groups can be selectively deprotected
using 2-5% of hydrazine in DMF. Deprotected side chains of Asp/Glu
(L- or D-forms) and Lys/Orn/Dab/Dap (L- or D-forms) can then be
coupled, e.g. by using the coupling reaction conditions described
above.
[0101] Peptide backbone amides may be N-methylated (i.e. alpha
amino methylated). This may be achieved by directly using
Fmoc-N-methylated amino acids during peptide synthesis.
Alternatively, N-methylation under Mitsunobu conditions may be
performed. First, a free primary amine group is protected using a
solution of 4-nitrobenzenesulfonyl chloride (Ns-CI) and
2,4,6-trimethylpyridine (collidine) in NMP. N-methylation may then
be achieved in the presence of triphenylphosphine, diisopropyl
azodicarboxylate (DIAD) and methanol. Subsequently, N-deprotection
may be performed using mercaptoethanol and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in NMP. For coupling
protected amino acids to N-methylated alpha amino groups, HATU,
HOAt and DIEA may be used.
[0102] The formation of the thioether (--S--) linkages (e.g. for
L.sup.1 or L.sup.2) can be achieved either on solid phase or in
solution phase. For example, the formation of thioether (--S--)
linkage can be achieved by coupling between a thiol-containing
compound (such as the thiol group on cysteine side chain) and an
alkyl halide (such as 3-(Fmoc-amino)propyl bromide and the like) in
an appropriate solvent (such as N,N-dimethylformamide and the like)
in the presence of base (such as N,N-diisopropylethylamine and the
like). If the reactions are carried out in solution phase, the
reactants used are preferably in equivalent molar ratio (1 to 1),
and the desired products can be purified by flash column
chromatography or high performance liquid chromatography (HPLC). If
the reactions are carried out on solid phase, meaning one reactant
has been attached to a solid phase, then the other reactant is
normally used in excess amount (>3 equivalents of the reactant
attached to the solid phase). After the reactions, the excess
unreacted reactant and reagents can be removed by sequentially
washing the solid phase (resin) using a combination of solvents,
such as N,N-dimethylformamide, methanol and dichloromethane, for
example.
[0103] The formation of the linkage (e.g. for L.sup.1 or L.sup.2)
between a thiol group and a maleimide group can be performed using
the conditions described above for the formation of the thioether
(--S--) linkage simply by replacing the alkyl halide with a
maleimide-containing compounds. Similarly, this reaction can be
conducted in solid phase or solution phase. If the reactions are
carried out in solution phase, the reactants used are preferably in
equivalent molar ratio (1 to 1), and the desired products can be
purified by flash column chromatography or high performance liquid
chromatography (HPLC). If the reactions are carried out on solid
phase, meaning one reactant has been attached to a solid phase,
then the other reactant is normally used in excess amount (>3
equivalents of the reactant attached to the solid phase). After the
reactions, the excess unreacted reactant and reagents can be
removed by sequentially washing the solid phase (resin) using a
combination of solvents, such as N,N-dimethylformamide, methanol
and dichloromethane, for example.
[0104] Non-peptide moieties (e.g. radiolabeling groups,
albumin-binding groups and/or linkers) may be coupled to the
peptide N-terminus while the peptide is attached to the solid
support. This is facile when the non-peptide moiety comprises an
activated carboxylate (and protected groups if necessary) so that
coupling can be performed on resin. For example, but without
limitation, a bifunctional chelator, such as
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
tris(tert-butyl ester) may be activated in the presence of
N-hydroxysuccinimide (NHS) and N,N'-dicyclohexylcarbodiimide (DCC)
for coupling to a peptide. Alternatively, a non-peptide moiety may
be incorporated into the compound via a copper-catalyzed click
reaction under either liquid or solid phase conditions.
Copper-catalyzed click reactions are well established in the art.
For example, 2-azidoacetic acid is first activated by NHS and DCC
and coupled to a peptide. Then, an alkyne-containing non-peptide
moiety may be clicked to the azide-containing peptide in the
presence of Cu.sup.2+ and sodium ascorbate in water and organic
solvent, such as acetonitrile (ACN) and DMF and the like.
Non-peptide moieties may also be added in solution phase, which is
routinely performed.
[0105] The synthesis of chelators is well-known and many chelators
are commercially available (e.g. from Sigma-Aldrich.TM./Milipore
Sigma.TM. and others). Protocols for conjugation of radiometals to
the chelators are also well known (e.g. see Example 1, below). The
synthesis of the silicon-fluorine-acceptor moieties can be achieved
following previously reported procedures (e.g. Bernard-Gauthier et
al. Biomed Res Int. 2014 2014:454503; Kostikov et al. Nature
Protocols 2012 7:1956-1963; Kostikov et al. Bioconjug Chem. 2012
18:23:106-114; each of which is incorporated by reference in its
entirety).
[0106] The synthesis or acquisition of radioisotope-substituted
aryl groups is likewise facile. The synthesis of the
R.sup.6R.sup.7BF.sub.3 component on the compounds can be achieved
following previously reported procedures (e.g.: Liu et al. Angew
Chem Int Ed 2014 53:11876-11880; Liu et al. J Nucl Med 2015
55:1499-1505; Liu et al. Nat Protoc 2015 10:1423-1432; Kuo et al.,
J Nucl Med 2019 60:1160-1166; each of which is incorporated by
reference in its entirety). Generally, the BF.sub.3-containing
motif can be coupled to the linker via click chemistry by forming a
1,2,3-triazole ring between a BF.sub.3-containing azido (or
alkynyl) group and an alkynyl (or azido) group on the linker, or by
forming an amide linkage between a BF.sub.3-containing carboxylate
and an amino group on the linker. To make the BF.sub.3-containing
azide, alkyne or carboxylate, a boronic acid ester-containing
azide, alkyne or carboxylate is first prepared following by the
conversion of the boronic acid ester to BF.sub.3 in a mixture of
HCl, DMF and KHF.sub.2. For alkyl BF.sub.3, the boronic acid
ester-containing azide, alkyne or carboxylate can be prepared by
coupling boronic acid ester-containing alkyl halide (such as
iodomethylboronic acid pinacol ester) with an amine-containing
azide, alkyne or carboxylate (such as N,N-dimethylpropargylamine).
For aryl BF.sub.3, the boronic acid ester can be prepared via
Suzuki coupling using aryl halide (iodine or bromide) and
bis(pinacolato)diboron.
[0107] .sup.18F-Fluorination of the BF.sub.3-containing compounds
via .sup.18F-.sup.19F isotope exchange reaction can be achieved
following previously published procedures (Liu et al. Nat Protoc
2015 10:1423-1432, incorporated by reference in its entirety).
Generally, .about.100 nmol of the BF.sub.3-containing compound is
dissolved in a mixture of 15 .mu.l of pyridazine-HCl buffer
(pH=2.0-2.5, 1 M), 15 .mu.l of DMF and 1 .mu.l of a 7.5 mM
KHF.sub.2 aqueous solution. .sup.18F-Fluoride solution (in saline,
60 .mu.l) is added to the reaction mixture, and the resulting
solution is heated at 80.degree. C. for 20 min. At the end of the
reaction, the desired product can be purified by solid phase
extraction or by reversed high performance liquid chromatography
(HPLC) using a mixture of water and acetonitrile as the mobile
phase.
[0108] When the peptide has been fully synthesized on the solid
support, the desired peptide may be cleaved from the solid support
using suitable reagents, such as TFA, tri-isopropylsilane (TIS) and
water. Side chain protecting groups, such as Boc,
pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trityl (Trt) and
tert-butyl (tBu) are simultaneously removed (i.e. deprotection).
The crude peptide may be precipitated and collected from the
solution by adding cold ether followed by centrifugation.
Purification and characterization of the peptides may be performed
by standard separation techniques, such as high performance liquid
chromatography (HPLC) based on the size, charge and polarity of the
peptides. The identity of the purified peptides may be confirmed by
mass spectrometry or other similar approaches.
[0109] The present invention will be further illustrated in the
following examples for the synthesis and evaluation of specific
compounds.
EXAMPLES
Experimental Methods and Procedures
[0110] Chemical Synthesis
[0111] Reagents and solvents were purchased from commercial sources
and used without further purification, unless otherwise stated.
High performance liquid chromatography (HPLC) was performed on 1)
an Agilent 1260 infinity system equipped with a model 1200
quaternary pump, a model 1200 UV absorbance detector and a Bioscan
NaI scintillation detector or 2) an Agilent 1260 Infinity II
Preparative System equipped with a model 1260 Infinity II
preparative binary pump, a model 1260 Infinity variable wavelength
detector (set at 220 nm), and a 1290 Infinity II preparative
open-bed fraction collector. The HPLC column used for purification
was a preparative column (Gemini, NX-C18, 5 .mu.m, 110 .ANG.,
50.times.30 mm) purchased from Phenomenex. The HPLC column used for
radiosynthesis was a Phenomenex Luna C18semi-preparative column
(5.mu., 250.times.10 mm) and for quality control was a Phenomenex
Luna C18 analytical column (5.mu., 250.times.4.6 mm). The
identities of peptides were confirmed by mass analysis using an AB
SCIEX 4000 QTRAP mass spectrometer system with an ESI ion source or
a Waters 2695 Separation module and Waters-Micromass ZQ mass
spectrometer system. A Bruker 300 Ultrashield NMR system was used
to obtain the .sup.1H, .sup.19F, .sup.11B, and .sup.13C NMR
Data.
[0112] Unless otherwise noted, amino acid couplings were performed
using 4/8/4 equivalents of the Fmoc-Amino Acid/DIC/Oxyma for 6 mins
at 90.degree. C. using the CEM Liberty Blue Microwave Peptide
Synthesizer. Fmoc groups were removed after amino acid couplings
were completed using a 20% piperidine solution in DMF for 1 min at
90.degree. C. unless otherwise noted. The resin was washed three
times with 3 mL DMF after each deprotection. Peptides were
deprotected and simultaneously cleaved from the resin using a
92.5/5/2.5 TFA/TIS/H.sub.2O cocktail unless otherwise stated.
[0113] Synthesis of BL02
[0114] The chemical structure of BL02 is below.
##STR00162##
[0115] Fmoc-Rink Amide ProTide resin (CEM, 0.25 mmol, 0.58 mmol/g)
was deprotected with 20% v/v piperidine in DMF for 1 min at
90.degree. C. twice. Fmoc-Lys(ivDde)-OH was then coupled to the
resin. The resin was then capped using 1-acetylimidazole in DMF
(0.1 w/v) at room temperature for 30 minutes. Fmoc-Lys(iPr,Boc)-OH,
Fmoc-D-Glu(OAII)-OH, Fmoc-Gly-OH (coupled twice), Fmoc-2NaI-OH
(coupled twice), Fmoc-D-Arg-OH (coupled twice for 4 mins each),
Fmoc-Lys(iPr,Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Phe-OH (coupled
twice) were sequentially coupled to the peptidyl resin. At a 0.1
mmol scale, the --OAllyl protecting group on D-Glu was removed
using Pd(PPh.sub.3).sub.4 (25 mg)/Phenylsilane (600 .mu.L) in DCM
(5 mL) (2.times.5 min at 35.degree. C.). The Na-Fmoc on Phe was
then removed, and cyclization was performed using DIC/HOBt in DMF
(3.times.10 min at 90.degree. C.). Following cyclization, the ivDde
protecting group was removed by 2% v/v hydrazine in DMF (5.times.5
min at RT). The resin (0.025 mmol) was coupled with three
Fmoc-Glu(OtBu)-OH sequentially. Afterwards, the chelator DOTA
tri-t-butyl ester (4 equiv.) in DMF was coupled to the terminal
amine with HATU/DIEA (4/8 equiv.) for 10 minutes at 50.degree. C.,
with two coupling cycles. The peptide was deprotected and cleaved
at 3.5 h at 35.degree. C. and the crude peptide mixture was
concentrated and precipitated in cold diethyl ether. The suspension
was centrifuged at 2500 RPM for 7 minutes, the supernatant diethyl
ether was discarded, and the solids were diluted into water, frozen
and lyophilized to yield a white powder. The reaction mixture was
purified by HPLC using the preparative column eluted with first
10-18% acetonitrile in water with 0.1% TFA for 0-16 mins, then
18-22% acetonitrile for 16-20 mins, then 22-25% acetonitrile in
20-25 mins at a flow rate of 30 mL/min. The retention time was 22.4
min, and the yield of the peptide was 9.0%. ESI-MS: calculated
[M+2H].sup.2+ for BL02 C.sub.99H.sub.147N.sub.23O.sub.27 1046.5508;
found [M+2H].sup.2+ 1046.2185.
[0116] Synthesis of Ga-BL02
[0117] For Ga-BL02, a solution of BL02 (1.89 mg, 0.90 .mu.mol) and
GaCl.sub.3 (0.8 mg, 4.5 .mu.mol) in 400 .mu.L sodium acetate buffer
(0.1 M, pH 4.2) was incubated at 70.degree. C. for 15 min. The
reaction mixture was purified by HPLC using the preparative column
eluted with first 10-18% acetonitrile in water with 0.1% TFA for
0-16 min, then 18-22% acetonitrile for 16-20 min, then 22-25%
acetonitrile in 20-25 min at a flow rate of 30 mL/min. The
retention time of Ga-BL02 was 23.2 min, and the yield of the
peptide was 80%. ESI-MS: calculated [M+2H].sup.2+ for Ga-BL02
C.sub.99H.sub.147GaN.sub.23O.sub.27 1080.0058; found [M+2H].sup.2+
1080.1585.
[0118] Synthesis of Lu-BL02
[0119] For Lu-BL02, a solution of BL02 (1.1 mg, 0.53 .mu.mol) and
LuCl.sub.3 (0.76 mg, 2.7 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 90.degree. C. for 20 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 13-33% acetonitrile in water with 0.1% TFA over
20 mins at a flow rate of 30 mL/min. The retention time of Lu-BL02
was 11.6 min, and the yield of the peptide was 42%. ESI-MS:
calculated [M+3H].sup.3+ for Lu-BL02
C.sub.99H.sub.148LuN.sub.23O.sub.27 755.6780; found [M+3H].sup.3+
755.0988.
[0120] Synthesis of BL03
[0121] The chemical structure of BL03 is below.
##STR00163##
[0122] From the synthesis of BL02, following the removal of the
ivDde group at a 0.025 mmol scale, Fmoc-Lys(ivDde)-OH,
Fmoc-Glu(OtBu)-OH, and 4-(p-iodophenyl)butyric acid were coupled
sequentially with using 4/8/4 equiv. of Fmoc-AA-OH/DIC/Oxyma in DMF
for 4 min at 90.degree. C. After each coupling, the Fmoc group was
removed with 20% v/v piperidine in DMF for 1 min at 90.degree. C.
and the resin washed three times. The ivDde protecting group was
then removed by 3% v/v hydrazine in DMF (5.times.5 min at RT). The
chelator DOTA tri-t-butyl ester (4 equiv.) in DMF was coupled twice
to the c-amine group on the Lys side-chain with HATU/DIEA (4/8
equiv.) for 10 min at 50.degree. C. The peptide was deprotected and
simultaneously cleaved from the resin by treating with a cocktail
solution of 92.5/5/2.5 TFA/TIS/H.sub.2O for 3 h at 35.degree. C.
The crude peptide mixture was worked up as previously described.
The reaction mixture was purified by HPLC using the preparative
column eluted with 15-33.75% acetonitrile in water with 0.1% TFA
for 0-25 mins at a flow rate of 30 mL/min. The retention time was
19.98 min, and the yield of the peptide was 6.7%. ESI-MS:
calculated [M+2H].sup.2+ for BL03
C.sub.105H.sub.156IN.sub.23O.sub.23 1117.5406; found [M+2H].sup.2+
1117.6880.
[0123] Synthesis of Lu-BL03
[0124] For Lu-BL03, a solution of BL03 (2.77 mg, 1.23 .mu.mol) and
LuCl.sub.3 (1.74 mg, 6.17 .mu.mol) in 400 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-30% acetonitrile in water with 0.1% TFA for
0-20 min at a flow rate of 30 mL/min. The retention time of Lu-BL03
was 19.11 min, and the yield of the peptide was 59%. ESI-MS:
calculated [M+3H].sup.3+ for Lu-BL03
C.sub.105H.sub.155LuN.sub.23O.sub.23 803.0045; found [M+3H].sup.3+
803.2280.
[0125] Synthesis of BL04
[0126] The chemical structure of BL04 is below.
##STR00164##
[0127] From the synthesis of BL02, following the coupling of the
triglutamate linker at a 0.025 mmol scale, Fmoc-Lys(ivDde)-OH,
Fmoc-Glu(OtBu)-OH and 2-azidoacetic acid were coupled on
sequentially. The ivDde protecting group was then removed by 2% v/v
hydrazine in DMF (5.times.5 min at RT), and Fmoc-Glu(OtBu)-OH and
2-azidoacetic acid were coupled on sequentially. The peptide was
deprotected and cleaved for 4 h at 35.degree. C. and the crude
peptide mixture was worked up as previously described. The reaction
mixture was purified by HPLC using the preparative column eluted
with 20-30% acetonitrile in water with 0.1% TFA for 0-15 mins at a
flow rate of 30 mL/min. The retention time was 10.19 min. The
fractions were collected and lyophilized. The yield of the peptide
was 5.2%. The azido precursor (0.825 mg, 0.37 .mu.mol) was
dissolved in 3 mL of H.sub.2O. 5 .mu.L of 1 M CuSO.sub.4, 5 .mu.L
of 1 M propargyl-AMBF.sub.3, 500 .mu.L of 0.1 M NH.sub.4OH
solution, and 6 .mu.L of 1 M sodium ascorbate were added
sequentially and heated to 45.degree. C. until the reaction mixture
turned clear and starting material was consumed based on HPLC. The
reaction mixture was purified again by HPLC using the preparative
column eluted with 10-30% acetonitrile in water with 0.1% formic
acid for 0-15 mins at a flow rate of 30 mL/min. The retention time
was 8.14 min and the yield of the peptide was 65%.
[0128] Synthesis of BL05
[0129] The chemical structure of BL05 is below.
##STR00165##
[0130] From the synthesis of BL02, following the removal of the
ivDde group at a 0.025 mmol scale, the resin containing the
macrocyclic peptide was coupled three times using 4/8/4 equiv. of
Fmoc-D-Arg(Pbf)-OH/DIC/Oxyma in DMF for 4 min at 90.degree. C.,
with two coupling cycles for each coupling. After each double
coupling, the Fmoc group was removed with 20% v/v piperidine in DMF
for 1 min at 90.degree. C. and the resin washed three times before
the next coupling. Afterwards, the chelator DOTA tri-t-butyl ester
(4 equiv.) in DMF was coupled to the terminal amine with HATU/DIEA
(4/8 equiv.) for 10 minutes at 50.degree. C., with two coupling
cycles. The peptide was deprotected and simultaneously cleaved from
the resin by treating with a cocktail solution of 92.5/5/2.5
TFA/TIS/H.sub.2O for 4.5 h at 40.degree. C. and the crude peptide
mixture was worked up as previously described. The reaction mixture
was purified by HPLC using the preparative column eluted with first
10-15% acetonitrile in water with 0.1% TFA for 0-5 mins, then 15%
acetonitrile for 5-10 mins, then 15-25% acetonitrile in 10-20 mins
at a flow rate of 30 mL/min. The retention time was 18.3 min, and
the yield of the peptide was 5.0%. ESI-MS: calculated [M+3H].sup.3+
for BL05 C.sub.102H.sub.165N.sub.32O.sub.21 725.0948; found
[M+3H].sup.3+ 725.5924.
[0131] Synthesis of Ga-BL05
[0132] For Ga-BL05, a solution of BL05 (1.0 mg, 0.46 .mu.mol) and
GaCl.sub.3 (0.56 mg, 3.2 .mu.mol) in 300 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with first 10-15% acetonitrile in water with 0.1% TFA
for 0-5 mins, then 15% acetonitrile for 5-10 mins, then 15-25%
acetonitrile in 10-20 mins at a flow rate of 30 mL/min. The
retention time of Ga-BL05 was 18.7 min, and the yield of the
peptide was 89%. ESI-MS: calculated [M+3H].sup.3+ for Ga-BL05
C.sub.102H.sub.163GaN.sub.32O.sub.21 747.3981; found [M+3H].sup.3+
747.6309.
[0133] Synthesis of BL06
[0134] The chemical structure of BL06 is below.
##STR00166##
[0135] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) containing the macrocyclic
peptide was coupled with Fmoc-Pip-OH/HATU/DIEA in DMF for 10 min at
50.degree. C. for two cycles. The chelator DOTA tri-t-butyl ester
(4 equiv.) in DMF was coupled to the terminal amine with HATU/DIEA
(4/8 equiv.) for 10 minutes at 50.degree. C., with two coupling
cycles. The peptide was deprotected and cleaved for 4 h at
35.degree. C. and the crude peptide mixture was worked up as
previously described. The reaction mixture was purified by HPLC
using the preparative column eluted with 10-25% acetonitrile in
water with 0.1% TFA for 0-15 mins at a flow rate of 30 mL/min. The
retention time was 14.0 min, and the yield of the peptide was 9.0%.
ESI-MS: calculated [M+2H].sup.2+ for BL06
C.sub.91H.sub.140N.sub.22O.sub.19 922.5327; found [M+2H].sup.2+
922.8853.
[0136] Synthesis of Ga-BL06
[0137] For Ga-BL06, a solution of BL06 (1.54 mg, 0.84 .mu.mol) and
GaCl.sub.3 (1.0 mg, 5.85 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 10-25% acetonitrile in water with 0.1% TFA for
0-15 min at a flow rate of 30 mL/min. The retention time of Ga-BL06
was 13.6 min, and the yield of the peptide was 87%. ESI-MS:
calculated [M+2H].sup.2+ for Ga-BL06
C.sub.91H.sub.138GaN.sub.22O.sub.19 955.9877; found [M+2H].sup.2+
956.8644.
[0138] Synthesis of Lu-BL07
[0139] The chemical structure of Lu-BL07 is below.
##STR00167##
[0140] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) containing the macrocyclic
peptide was coupled with three Fmoc-Glu(OtBu)-OH, a
Fmoc-Lys(ivDde)-OH and a Fmoc-Glu(OtBu)-OH sequentially.
2-Azidoacetic acid was then coupled for 10 min at 90.degree. C.,
with two cycles. The ivDde protecting group was then removed by 2%
v/v hydrazine in DMF (5.times.5 min at RT). The peptide was
deprotected and cleaved for 4 h at 35.degree. C. and the crude
peptide mixture was worked up as previously described and purified
by HPLC. The fractions were collected and lyophilized and dissolved
in 3 mL of H.sub.2O. 5 uL of 1 M CuSO.sub.4,5 uL of 1 M
propargyl-AMBF.sub.3, 500 uL of 0.1 M NH.sub.4OH solution, and 6 uL
of 1 M sodium ascorbate were added sequentially and heated to
45.degree. C. until the reaction mixture turned clear and starting
material was consumed based on HPLC. The reaction mixture was
purified by HPLC and the fractions collected and lyophilized. The
chelator DOTA NHS-ester (0.93 mg, 1.22 .mu.mol) in DMF and DIEA
(0.72 .mu.L, 4.1 umol), was coupled to the terminal amine of the
peptide (0.9 mg, 0.41 .mu.mol). After completion of the reaction in
3 hours as determined by HPLC, the reaction mixture was diluted in
water and purified via preparative HPLC. The reaction yield was
74%. To the unchelated peptide (0.65 mg, 0.23 .mu.L), LuCl.sub.3
(0.28 mg, 1 .mu.mol) was added in 500 .mu.L sodium acetate buffer
(0.1 M, pH 4.2) and incubated at 90.degree. C. for 15 min. The
reaction mixture was purified by HPLC using the preparative column
eluted with 5-25% acetonitrile in water with 0.1% formic acid for
0-20 mins at a flow rate of 30 mL/min. The retention time was 13.9
min, and the overall yield of the peptide was 1.4%. ESI-MS:
calculated [M+3H].sup.3+ for Lu-BL07
C.sub.118H.sub.179BF.sub.3LuN.sub.30O.sub.32 923.7579; found
[M+3H].sup.3+ 923.2525.
[0141] Synthesis of BL08
[0142] The chemical structure of BL08 is below.
##STR00168##
[0143] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) was coupled with three
Fmoc-Glu(OtBu)-OH sequentially. After the final Fmoc deprotection,
the resin washed three times before the next coupling. The resin
was placed into a spin column and was swelled using degassed and
freshly distilled DMF (10 mL) for 30 minutes. The solution was then
drained and rinsed with DCM. At a 0.025 mmol scale, PepBF3 JL3 (see
below) (32 mg, 149 .mu.mol) was dissolved in DMF (5 mL) and was
transferred to the spin column. HBTU (54.5 mg, 144 .mu.mol) was
directly added to the bead solution followed by DIPEA (52 .mu.L,
609 .mu.mol). The mixture was mixed for 4 hours using a tube
rotator. The solution was drained and rinsed with DCM, DMF, and DCM
three times in 10 mL portions each and dried in vacuo for 16 hours.
The dried beads were transferred into a falcon tube and were
suspended in 500 .mu.L DCM and added with 50 .mu.L TIPS, 10 .mu.L
H.sub.2O, and a stir bar. KHF.sub.2 (200 mg) was placed into a
separate falcon tube. TFA (1 mL) was added to the falcon tube using
a hypodermic needle and 1 mL syringe. The tube was then sealed and
sonicated until all the solids were observed to completely
dissolve. After complete dissolution, the mixture was added to the
falcon tube containing the beads. The mixture was stirred uncapped
for 1 hour. Afterwards, the mixture was cooled then diluted with
H.sub.2O (1 mL) in an ice bath followed by the slow addition of
excess NH.sub.4OH until basic. ACN was then added to the mixture
and the solution was filtered and concentrated at low heat. The
resulting mixture was diluted into water, frozen, and lyophilized
to yield a white powder. This was then triturated with ACN and
centrifuged. The supernatant was collected and concentrated to
yield the crude peptide mixture which was purified by HPLC using
the preparative column, eluted with 10-20% acetonitrile in water
with 0.1% formic acid over 15 mins at a flow rate of 30 mL/min. The
retention time was 9.12 min, and the yield of BL08 was 5.8%.
ESI-MS: calculated [M+2H].sup.2+ for
C.sub.90H.sub.136BF.sub.3N.sub.20O.sub.21 950.5112, found
950.6130.
[0144] Synthesis of BL09
[0145] The chemical structure of BL09 is below.
##STR00169##
[0146] From the synthesis of BL08, following the removal of the
Fmoc group after the third coupling of Fmoc-Glu(OtBu)-OH,
2-Azidoacetic acid was coupled for 10 min at 90.degree. C., with
two cycles. The peptide was deprotected and cleaved for 3 h at
35.degree. C. and the crude peptide mixture was worked up as
previously described. The reaction mixture was purified by HPLC
using the preparative column eluted with 18-28% acetonitrile in
water with 0.1% TFA over 15 mins at a flow rate of 30 mL/min. The
retention time was 11.87 min and the yield of the peptide was
11.2%. The fractions were collected and lyophilized and dissolved
in 3 mL of H.sub.2O. 5 uL of 1 M CuSO.sub.4, 5 uL of 1 M
propargyl-AMBF.sub.3, 500 uL of 0.1 M NH.sub.4OH solution, and 6 uL
of 1 M sodium ascorbate were added sequentially and heated to
45.degree. C. until the reaction mixture turned clear and starting
material was consumed based on HPLC. The reaction mixture was
purified again by HPLC using the preparative column eluted with
10-20% acetonitrile in water with 0.1% formic acid over 15 mins at
a flow rate of 30 mL/min. The retention time was 8.73 min and the
yield of BL09 was 47%. ESI-MS: calculated [M+2H].sup.+2
C.sub.91H.sub.135BF.sub.3N.sub.23O.sub.21 977.0119, found
977.1859.
[0147] Synthesis of BL17
[0148] The chemical structures of BL17, BL20 and BL25 are
below.
##STR00170## ##STR00171##
[0149] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) was coupled with three
Fmoc-Aad(OtBu)-OH sequentially. Afterwards, the chelator DOTA
tri-t-butyl ester (4 equiv.) in DMF was coupled to the terminal
amine with HATU/DIEA (4/8 equiv.) for 18 hours at room temperature.
The peptide was deprotected and cleaved for 3.5 h at 35.degree. C.
and the crude peptide mixture was worked up as previously
described. The reaction mixture was purified by HPLC using the
preparative column eluted with 10-30% acetonitrile in water with
0.1% TFA over 20 mins at a flow rate of 30 mL/min. The retention
time was 14.3 min and the yield of the peptide was 7.2%. ESI-MS:
calculated [M+2H].sup.2+ for BL17
C.sub.102H.sub.155N.sub.23O.sub.27 1067.5743; found [M+2H].sup.2+
1067.4061.
[0150] Synthesis of Ga-BL17
[0151] For Ga-BL17, a solution of BL17 (2.3 mg, 1.1 .mu.mol) and
GaCl.sub.3 (0.95 mg, 5.4 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 90.degree. C. for 20 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 10-30% acetonitrile in water with 0.1% TFA over
20 mins at a flow rate of 30 mL/min. The retention time of Ga-BL17
was 14.6 min, and the yield of the peptide was 86%. ESI-MS:
calculated [M+2H].sup.2+ for Ga-BL17
C.sub.102H.sub.153GaN.sub.23O.sub.27 1101.0292; found [M+2H].sup.2+
1100.9840.
[0152] Synthesis of BL18
[0153] The chemical structure of BL18 is below.
##STR00172##
[0154] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) was coupled with Glu(OtBu),
Glu(OtBu) and Lys(ivDde) sequentially. Afterwards, the chelator
DOTA tri-t-butyl ester (4 equiv.) in DMF was coupled to the
terminal amine with HATU/DIEA (4/8 equiv.) for 18 hours at room
temperature. The ivDde protecting group was removed by 2% v/v
hydrazine in DMF (5.times.5 min at RT). Fmoc-Gly-OH was then
coupled. Afterwards, following removal of the Fmoc group,
4-(p-iodophenyl)butyric acid (4 equiv.) was coupled using HATU and
DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. The peptide
was deprotected and cleaved for 3.5 h at 35.degree. C. and the
crude peptide mixture was worked up as previously described. The
reaction mixture was purified by HPLC using the preparative column
eluted with 20-40% acetonitrile in water with 0.1% TFA over 15 mins
at a flow rate of 30 mL/min. The retention time was 9.1 min, and
the yield of the peptide was 4.0%. ESI-MS: calculated [M+3H].sup.3+
for BL18 C.sub.112H.sub.167N.sub.25O.sub.27 807.3842; found
[M+3H].sup.2+ 807.1577.
[0155] Synthesis of Lu-BL18
[0156] For Lu-BL18, a solution of BL18 (1.8 mg, 0.74 .mu.mol) and
LuCl.sub.3 (1.0 mg, 3.6 .mu.mol) in 500 .mu.L sodium acetate buffer
(0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min. The
reaction mixture was purified by HPLC using the preparative column
eluted with 20-40% acetonitrile in water with 0.1% TFA over 15 mins
at a flow rate of 30 mL/min. The retention time of Lu-BL17 was 9.3
min, and the yield of the peptide was 75%.
[0157] Synthesis of BL19
[0158] The chemical structure of BL19 is below.
##STR00173##
[0159] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) was coupled with Glu(OtBu),
Lys(ivDde) and Glu(OtBu) sequentially. Afterwards, the chelator
DOTA tri-t-butyl ester (4 equiv.) in DMF was coupled to the
terminal amine with HATU/DIEA (4/8 equiv.) for 18 hours at room
temperature. The ivDde protecting group was removed by 2% v/v
hydrazine in DMF (5.times.5 min at RT). Fmoc-Gly-OH was then
coupled. Afterwards, following removal of the Fmoc group,
4-(p-iodophenyl)butyric acid (4 equiv.) was coupled using HATU and
DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. The peptide
was deprotected and cleaved for 3.5 h at 35.degree. C. and the
crude peptide mixture was worked up as previously described. The
reaction mixture was purified by HPLC using the preparative column
eluted with 20-40% acetonitrile in water with 0.1% TFA over 15 mins
at a flow rate of 30 mL/min. The retention time was 9.1 min, and
the yield of the peptide was 4.6%. ESI-MS: calculated [M+3H].sup.3+
for BL19 C.sub.112H.sub.167N.sub.25O.sub.27 807.3842; found
[M+3H].sup.3+ 807.3602.
[0160] Synthesis of Lu-BL19
[0161] For Lu-BL19, a solution of BL19 (1.34 mg, 0.55 .mu.mol) and
LuCl.sub.3 (0.78 mg, 2.76 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time of Lu-BL19
was 9.3 min, and the yield of the peptide was 71%. ESI-MS:
calculated [M+3H].sup.3+ for Lu-BL19
C.sub.112H.sub.165ILuN.sub.25O.sub.27 865.0259; found [M+3H].sup.3+
864.5719.
[0162] Synthesis of BL20
[0163] The chemical structure of BL20 is shown above.
[0164] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) was coupled with three
Fmoc-D-Glu(OtBu)-OH sequentially. Afterwards, the chelator DOTA
tri-t-butyl ester (4 equiv.) in DMF was coupled to the terminal
amine with HATU/DIEA (4/8 equiv.) for 18 hours at room temperature.
The peptide was deprotected and cleaved for 3.5 h at 35.degree. C.
and the crude peptide mixture was worked up as previously
described. The reaction mixture was purified by HPLC using the
preparative column eluted with 11-31% acetonitrile in water with
0.1% TFA over 20 mins at a flow rate of 30 mL/min. The retention
time was 13.3 min, and the yield of the peptide was 5.9%. ESI-MS:
calculated [M+2H].sup.2+ for BL20 C.sub.99H.sub.147N.sub.23O.sub.27
1046.5508; found [M+2H].sup.2+ 1045.9112.
[0165] Synthesis of Ga-BL20
[0166] For Ga-BL20, a solution of BL20 (0.94 mg, 0.45 .mu.mol) and
GaCl.sub.3 (0.46 mg, 2.6 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 11-31% acetonitrile in water with 0.1% TFA for
30 min at a flow rate of 30 mL/min. The retention time of Ga-BL20
was 13.4 min, and the yield of the peptide was 85%. ESI-MS:
calculated [M+2H].sup.2+ for Ga-BL20
C.sub.99H.sub.147GaN.sub.23O.sub.27 1080.0058; found [M+2H].sup.2+
1079.3370.
[0167] Synthesis of BL21
[0168] The chemical structure of BL21 is below.
##STR00174##
[0169] From the synthesis of BL19, following the removal of the
second ivDde group, Fmoc-Gly-OH and Fmoc-NH-PEG.sub.4-COOH and was
coupled sequentially. Afterwards, following removal of the Fmoc
group, 4-(p-iodophenyl)butyric acid (4 equiv.) was coupled using
HATU and DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. The
peptide was deprotected and cleaved for 3.5 h at 35.degree. C. and
the crude peptide mixture was worked up as previously described.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time was 9.5
min, and the yield of the peptide was 6.1%.
[0170] Synthesis of Lu-BL21
[0171] For Lu-BL21, a solution of BL21 (1.51 mg, 0.57 .mu.mol) and
LuCl.sub.3 (0.80 mg, 2.82 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time of Lu-BL21
was 9.9 min, and the yield of the peptide was 97%.
[0172] Synthesis of BL22
[0173] The chemical structures of BL22, BL23, BL26, BL27, BL28 and
BL29 are below.
##STR00175##
[0174] From the synthesis of BL19, following the coupling of
Fmoc-Gly-OH, the Fmoc group was removed and
4-(p-chlorophenyl)butyric acid (4 equiv.) was coupled using HATU
and DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. with two
cycles. The peptide was deprotected and cleaved for 3.5 h at
35.degree. C. and the crude peptide mixture was worked up as
previously described. The reaction mixture was purified by HPLC
using the preparative column eluted with 20-40% acetonitrile in
water with 0.1% TFA for 0-15 min at a flow rate of 30 mL/min. The
retention time was 8.2 min, and the yield of the peptide was 8.2%.
ESI-MS: calculated [M+2H].sup.2+ for BL22
C.sub.112H.sub.116ClN.sub.25O.sub.27 1164.6048; found [M+2H].sup.2+
1164.7199.
[0175] Synthesis of Ga-BL22
[0176] For Ga-BL22, a solution of BL22 (1.41 mg, 6.0 .mu.mol) and
GaCl.sub.3 (0.53 mg, 3.0 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA for
0-15 min at a flow rate of 30 mL/min. The retention time was 8.4
min, and the yield of the peptide was 75%. ESI-MS: calculated
[M+3H].sup.3+ for Ga-BL22 C.sub.112H.sub.166CIGaN.sub.25O.sub.27
799.3782; found [M+3H].sup.3+ 799.0323.
[0177] Synthesis of BL23
[0178] The chemical structure of BL23 is above.
[0179] From the synthesis of BL19, following the coupling of
Fmoc-Gly-OH, the Fmoc group was removed and
4-(4-methoxyphenyl)butyric acid (4 equiv.) was coupled using HATU
and DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. with two
cycles. The peptide was deprotected and cleaved for 3.5 h at
35.degree. C. and the crude peptide mixture was worked up as
previously described. The reaction mixture was purified by HPLC
using the preparative column eluted with 20-40% acetonitrile in
water with 0.1% TFA for 0-15 min at a flow rate of 30 mL/min. The
retention time was 7.0 min, and the yield of the peptide was 7.9%.
ESI-MS: calculated [M+3H].sup.3+ for BL23
C.sub.113H.sub.170N.sub.25O.sub.28 775.4221; found [M+3H].sup.3+
775.4712.
[0180] Synthesis of Ga-BL23
[0181] For Ga-BL23, a solution of BL23 (0.91 mg, 0.39 .mu.mol) and
GaCl.sub.3 (0.33 mg, 1.89 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA for
0-15 min at a flow rate of 30 mL/min. The retention time was 7.8
min, and the yield of the peptide was 98%.
[0182] Synthesis of Lu-BL23
[0183] For Lu-BL23, a solution of BL23 (0.80 mg, 0.34 .mu.mol) and
LuCl.sub.3 (0.47 mg, 1.67 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time was 7.5
min, and the yield of the peptide was 84%.
[0184] Synthesis of BL24
[0185] The chemical structure of BL24 is below.
##STR00176##
[0186] From the synthesis of BL19, following the removal of the
second ivDde group, Fmoc-Glu(OtBu)-OH was coupled. Afterwards,
following removal of the Fmoc group, 4-(p-iodophenyl)butyric acid
(4 equiv.) was coupled using HATU and DIEA (4 and 8 equiv.) for 10
minutes at 50.degree. C. The peptide was deprotected and cleaved
for 3.5 h at 35.degree. C. and the crude peptide mixture was worked
up as previously described. The reaction mixture was purified by
HPLC using the preparative column eluted with 20-40% acetonitrile
in water with 0.1% TFA over 15 mins at a flow rate of 30 mL/min.
The retention time was 8.9 min, and the yield of the peptide was
6.1%.
[0187] Synthesis of Ga-BL24
[0188] For Ga-BL24, a solution of BL24 (0.95 mg, 0.38 .mu.mol) and
GaCl.sub.3 (0.34 mg, 1.94 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time was 9.3
min, and the yield of the peptide was 86%.
[0189] Synthesis of BL25
[0190] The chemical structure of BL25 is shown above.
[0191] From the synthesis of BL02, following the removal of the
ivDde group, the resin (0.025 mmol) was coupled with three
Fmoc-D-Asp(OBno)-OH sequentially using a 2/4/2 equiv. of amino
acid/DIC/Oxyma. The Fmoc was deprotected at room temperature for 5
minutes between couplings. Afterwards, the chelator DOTA
tri-t-butyl ester (4 equiv.) in DMF was coupled to the terminal
amine with HATU/DIEA (4/8 equiv.) for 18 hours at room temperature.
The peptide was deprotected and cleaved for 3.5 h at 35.degree. C.
and the crude peptide mixture was worked up as previously
described. The reaction mixture was purified by HPLC using the
preparative column eluted with 12-32% acetonitrile in water with
0.1% TFA for 0-20 min at a flow rate of 30 mL/min. The retention
time was 12.0 min, and the yield of the peptide was 5.3%. ESI-MS:
calculated [M+2H].sup.2+ for BL25 C.sub.96H.sub.143N.sub.23O.sub.27
1025.5273; found [M+2H].sup.2+ 1024.9492.
[0192] Synthesis of Ga-BL25
[0193] For Ga-BL25, a solution of BL25 (2.06 mg, 1.0 .mu.mol) and
GaCl.sub.3 (0.97 mg, 5.55 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 12-32% acetonitrile in water with 0.1% TFA for
0-20 min at a flow rate of 30 mL/min. The retention time was 12.3
min, and the yield of the peptide was 76%. ESI-MS: calculated
[M+3H].sup.3+ for Ga-BL25 C.sub.96H.sub.143GaN.sub.23O.sub.27
706.6599; found [M+3H].sup.3+ 706.1981.
[0194] Synthesis of BL26
[0195] The chemical structure of BL26 is shown above.
[0196] From the synthesis of BL19, the coupling of Fmoc-Gly-OH, the
Fmoc group was removed and 1,18-octadecanedioic acid
mono-tert-butyl ester (4 equiv.) was coupled using HATU and DIEA (4
and 8 equiv.) for 10 minutes at 50.degree. C. with two cycles. The
peptide was deprotected and cleaved for 3.5 h at 35.degree. C. and
the crude peptide mixture was worked up as previously described.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time was 13.5
min, and the yield of the peptide was 10.5%.
[0197] Synthesis of Ga-BL26
[0198] For Ga-BL26, a solution of BL26 (1.43 mg, 0.57 .mu.mol) and
GaCl.sub.3 (4.8 mg, 2.75 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 22-44% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time was 12.5
min, and the yield of the peptide was 92%.
[0199] Synthesis of BL27
[0200] The chemical structure of BL27 is shown above.
[0201] From the synthesis of BL19, following the coupling of
Fmoc-Gly-OH, the Fmoc group was removed and
4-(4-fluorophenyl)butyric acid (4 equiv.) was coupled using HATU
and DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. with two
cycles. The peptide was deprotected and cleaved for 3.5 h at
35.degree. C. and the crude peptide mixture was worked up as
previously described. The reaction mixture was purified by HPLC
using the preparative column eluted with 20-40% acetonitrile in
water with 0.1% TFA over 15 mins at a flow rate of 30 mL/min. The
retention time was 7.8 min, and the yield of the peptide was
9.7%.
[0202] Synthesis of Ga-BL27
[0203] For Ga-BL27, a solution of BL27 (0.98 mg, 0.41 .mu.mol) and
GaCl.sub.3 (0.35 mg, 2.1 .mu.mol) in 500 .mu.L sodium acetate
buffer (0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min.
The reaction mixture was purified by HPLC using the preparative
column eluted with 20-40% acetonitrile in water with 0.1% TFA over
15 mins at a flow rate of 30 mL/min. The retention time was 7.6
min, and the yield of the peptide was 91%.
[0204] Synthesis of BL28
[0205] The chemical structure of BL28 is shown above.
[0206] From the synthesis of BL19, following the coupling of
Fmoc-Gly-OH, the Fmoc group was removed and
4-(4-methylphenyl)butyric acid (4 equiv.) was coupled using HATU
and DIEA (4 and 8 equiv.) for 10 minutes at 50.degree. C. with two
cycles. The peptide was deprotected and cleaved for 3.5 h at
35.degree. C. and the crude peptide mixture was worked up as
previously described. The reaction mixture was purified by HPLC
using the preparative column eluted with 20-40% acetonitrile in
water with 0.1% TFA over 15 mins at a flow rate of 30 mL/min. The
retention time was 7.9 min, and the yield of the peptide was
9.3%.
[0207] Synthesis of Ga-BL28
[0208] For Ga-BL28, a solution of BL28 (1.1 mg, 0.46 .mu.mol) and
GaCl.sub.3 (4.0 mg, 2.3 .mu.mol) in 500 .mu.L sodium acetate buffer
(0.1 M, pH 4.2) was incubated at 80.degree. C. for 15 min. The
reaction mixture was purified by HPLC using the preparative column
eluted with 20-40% acetonitrile in water with 0.1% TFA over 15 mins
at a flow rate of 30 mL/min. The retention time was 8.0 min, and
the yield of the peptide was 72%.
[0209] Synthesis of BL29
[0210] The chemical structure of BL29 is shown above.
[0211] From the synthesis of BL19, following the coupling of
Fmoc-Gly-OH, the Fmoc group was removed and 4-phenylbutyric acid (4
equiv.) was coupled using HATU and DIEA (4 and 8 equiv.) for 10
minutes at 50.degree. C. with two cycles. The peptide was
deprotected and cleaved for 3.5 h at 35.degree. C. and the crude
peptide mixture was worked up as previously described. The reaction
mixture was purified by HPLC using the preparative column eluted
with 20-40% acetonitrile in water with 0.1% TFA over 15 mins at a
flow rate of 30 mL/min. The retention time was 7.1 min, and the
yield of the peptide was 7.0%.
[0212] Chemical Synthesis of the PepBF.sub.3 Synthon
[0213] Synthesis of PepBF.sub.3 JL3
##STR00177##
[0214] 4-Dimethylamino-butyric acid benzyl ester (JL1). A round
bottom flask charged with .gamma.-aminobutyric acid (2 g, 19.4
mmol, 1 equiv.), formaldehyde (9 mL, 37% in solution v/v, 121 mmol,
6 equiv.), and formic acid (6 mL, 90% in solution, 143 mmol, 7
equiv.) and was stirred at 80.degree. C. for 48 h. The reaction was
monitored by TLC (10% MeOH in DCM, R.sub.f of intermediate=0.45,
stained with bromocresol green). The reaction mixture was cooled to
room temperature and HCl (6 mL, 4 M, 24.4 mmol, 1.25 equiv.) was
added. The reaction solution was dried down by rotary evaporation
to give a yellow solid intermediate 4-Dimethylamino-butyric acid to
which, benzyl alcohol (10 mL, 100 mmol, 5 equiv.), and
4-toluenesuphonic acid monohydrate (3.5 g, 20.9 mmol, 1.05 equiv.)
was added. The reaction was refluxed at 90.degree. C. for 2 h and
the reaction solution was cooled to room temperature. The toluene
phase was extracted with H.sub.2O (4.times.100 mL) and NaOH (1 M)
was added to the pooled aqueous phase until basic. The aqueous
phase was then extracted with EtOAc (3.times.100 mL). The EtOAc
extract was then washed with brine, dried with MgSO.sub.4,
filtered, and evaporated to give 3.31 g of crude orange oil which
was then purified via silica chromatography by using basified
silica and saturating the column in pet ether (PE) and the
fractions monitored by TLC (10% MeOH in DCM, R.sub.f of
intermediate=0.3). The compound was loaded directly onto the
silican and rinsed with PE, elutions were performed at 5 column
volumes (CV) of PE, 1:1; PE:EtOAc, EtOAc, DCM, 10% MeOH in DCM to
give JL1 in good yields (2.332 g, 52% over two steps). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. (ppm): 7.37 (m, 5H), 5.14 (s, 2H),
2.42 (t, 2H), 2.35 (t, 2H), 2.26 (s, 6H), 1.85 (quint, 2H).
##STR00178##
[0215]
(3-Benzyloxycarbonyl-propyl)-dimethyl-ammonium-methylenetrifluorobo-
rate (JL2). 4-Dimethylamino-butyric acid benzyl ester JL1 (2.5 g,
11.2 mmol) was dissolved in Et.sub.2O (50 mL) and DCM (50 mL).
Iodomethyl-boronylpinacolate (1.92 mL, 10.64 mmol, 0.95 equiv.) was
added drop-wise to the stirring mixture and the solution as allowed
to stir for 2 hours and was then placed into a 50.degree. C. bath
and stirred for 30 hours. The reaction was cooled and the solvents
were removed by rotovap to yield thick orange oil. The oil was
dissolved in ACN (100 mL) and diluted with 50 mL of water and was
combined with an aqueous solution of AgNO.sub.3 (100 mL, 0.18 M,
1.5 equiv.), then brine (0.12 mL, 0.71 mmol, 1.5 equiv.), producing
yellow precipitates and white precipitates respectively. The
mixture was filtered over celite and concentrated by rotovap. The
resulting white solids were triturated with ACN (100 mL), sonicated
for 30 mins, filtered through celite, and the filtrate was
concentrated to 15 mL and transferred to a plastic bottle. To
fluorinate, KHF.sub.2 (14 mL, 4 M, 112 mmol, 10 equiv.) and HCl (37
mL, 3 M, 112 mmol, 10 equiv.) were added to the solution. The
reaction was allowed to fluorinate for 1 hour and was quenched by
adding concentrated NH.sub.4OH until basic. The mixture was frozen
and lyophilized to give white solids that were extracted with
acetone (3.times.300 mL), and the pooled extract was dried down by
rotary evaporation to give the crude product as white solids which
was purified by silica chromatography. Column purification was
performed by using basified silica and saturating the column in DCM
and the fractions monitored by TLC (40% ACN in DCM, Rf=0.4). The
compound was dissolved with MeOH and was dry loaded onto silica.
The silica bound compound was placed into the column, and gradient
elutions were performed at 5 CVs of 0, 5, 10, 20% ACN in DCM to
yield the pure compound JL2 in good yields (2.7655 g, 86% over two
steps). .sup.1H NMR (300 MHz, ACN) .delta. (ppm): 7.37 (m, 5H),
5.13 (s, 2H), 3.30 (m, 2H), 3.03 (s, 6H), 2.48 (t, 2H), 2.45 (m,
2H), 2.09 (m, 2H). .sup.19F NMR (300 MHz, ACN) .delta. (ppm):
-141.02. .sup.11B NMR (300 MHz, ACN) .delta. (ppm): -2.09.
##STR00179##
[0216]
(3-Carboxy-propyl)-dimethyl-ammonium-methylenetrifluoroborate
(JL3).
(3-Benzyloxycarbonyl-propyl)-dimethyl-ammonium-methylenetrifluorob-
orate JL2 (2.65 g, 8.7 mmol) was placed in a round bottom flask and
the moisture was evacuated under reduced pressure and a warm water
bath. Argon was flushed through the flask and freshly distilled THE
(100 mL) was added into the flask and sonicated to achieve
dissolution. Palladium on charcoal (1.4 g, 10% Pd/C, 0.50 mmol,
0.11 equiv.) was added to the reaction vessel. The flask was capped
and stirred under H.sub.2(g) for 16 hours. Reaction progress was
monitored with TLC (40% ACN in DCM, Rf=0.4, UV, HBQ and BCG stain
active). Upon complete consumption of starting material, the
mixture was filtered through celite to remove the charcoal and
washed with methanol (3.times.50 mL). The solution was dried down
by rotary evaporation to give white solids (1.095 g, 60% overall
yield). .sup.1H NMR (300 MHz, D.sub.2O) .delta. (ppm): 3.21 (m,
2H), 2.97 (s, 6H), 2.42 (m, 2H), 2.39 (t, 2H), 2.08 (m, 2H).
.sup.19F NMR (300 MHz, D.sub.2O) .delta. (ppm): -140.91. .sup.11B
NMR (300 MHz, D20) .delta. (ppm): 2.08. .sup.13C NMR (300 MHz,
MeOD) .delta. (ppm): 174.26, 65.69, 52.37, 29.85, 18.19. ESI-MS
(-):C.sub.7H.sub.15BF.sub.3NO.sub.2 calculated exact mass 213.11
m/z; found [2M-H]-=425.2 m/z.
##STR00180##
[0217]
[3-(2-Hydroxy-ethylcarbamoyl)-propyl]-dimethyl-ammonium-methylenetr-
ifluoroborate (JL4).
(3-Carboxy-propyl)-dimethyl-ammonium-methylenetrifluoroborate JL3
(50 mg, 0.23 mmol) was charged into a round bottom flask and was
dissolved in DMF (5 mL). 3-aminopropanol (19.8 .mu.L, 0.25 mmol,
1.1 equiv.) was added into the mixture followed by the addition of
HBTU (97.9 mg, 0.25 mmol, 1.1 equiv.) and DIPEA (61 .mu.L, 0.35
mmol, 1.5 equiv.) and was allowed to stir for 16 hours. The
reaction was monitored using TLC (10% MeOH in DCM, Rf=0.2, product
is KMnO.sub.4 and HBQ stain active). Upon complete consumption of
the starting material, the reaction was dried down and was purified
by silica chromatography. Column purification was performed by
using basified silica and saturating the column in DCM. The crude
mixture was dissolved with MeOH and was dry loaded onto silica. The
silica bound compound was placed into the column, and gradient
elutions were performed at 5 CVs of 0, 5 and 10% MeOH in DCM to
yield the pure compound JL4 (8 mg, 13%). .sup.1H NMR (300 MHz,
D.sub.2O) .delta. (ppm): 3.60 (t, 2H), 3.29 (m, 4H), 3.06 (s, 6H),
2.43 (m, 2H), 2.28 (t, 2H), 2.08 (m, 2H), 1.73 (quint, 2H).
.sup.19F NMR (300 MHz, D20) .delta. (ppm): -141.23. .sup.11B NMR
(300 MHz, D20) .delta. (ppm): 2.05.
[0218] Radiochemical Synthesis
[0219] .sup.18F-Labeling: No-carrier-added [.sup.18F]fluoride was
obtained by bombardment of H.sub.2.sup.18O with 18-MeV protons
(Advanced Cyclotron Systems Inc) followed by trapping on an anion
exchange resin column (pre-activated with brine and washed with DI
water, without HCO.sub.3.sup.- preconditioning). The
[.sup.18F]fluoride was then eluted from the column using
HCl-pyridazine buffer (pH 2.0). Unlabeled trifluoroborate
precursors (100 nmol) was suspended in DMF (15 .mu.L). The eluted
[.sup.18F]fluoride (30-100 GBq) was added into a reaction vessel
containing the solution of BL08 or BL09. The vial was heated at
80.degree. C. for 20 minutes on a heating block and quenched upon
the addition of 1 mL of water. .sup.31,32 The mixture was purified
by semi-prep HPLC and quality control was performed via analytical
HPLC with the co-injection of the unlabeled standard with a
one-twelfth of the radiotracer. Radiochemical yields
(decay-corrected) were >10% and radiochemical purities were
>95%.
[0220] .sup.68Ga-Labeling: [.sup.68Ga]GaCl.sub.3 was eluted from an
iThemba Labs generator with a total of 4 mL of 0.1 M HCl. The
eluted [.sup.68Ga]GaCl.sub.3 solution was added to 2 mL of
concentrated HCl. This radioactive mixture was then added to a DGA
resin column and washed with 3 mL of 5 M HCl. The column was then
dried with air and the [.sup.68Ga]GaCl.sub.3 (0.10-0.50 GBq) was
eluted with 0.5 mL of water into a vial containing a solution of
the unlabeled precursor (25 .mu.g) in 0.7 mL HEPES buffer (2 M, pH
5.3). The reaction mixture was heated in a microwave oven (Danby;
DMW7700WDB) for 1 min at power setting 2. The mixture was purified
by semi-prep HPLC and quality control was performed via analytical
HPLC with the co-injection of the unlabeled standard with a
one-twelfth of the radiotracer. Radiochemical yields
(decay-corrected) were >50% and radiochemical purities were
>95%.
[0221] .sup.177Lu-Labeling: [.sup.177Lu]LuCl.sub.3 was purchased
from ITM Isotopen Technologien Munchen AG. [.sup.177Lu]LuCl.sub.3
(100-1000 MBq) in 0.04 M HCl (10-100 .mu.L) was added to a solution
of the unlabeled precursor (25 .mu.g) in 0.5 mL of NaOAc buffer
(0.1 M, pH 4.5). The reaction mixture was incubated at 100.degree.
C. for 15 min. The mixture was purified by semi-prep HPLC and
quality control was performed via analytical HPLC with the
co-injection of the unlabeled standard with a one-twelfth of the
radiotracer. Radiochemical yields (decay-corrected) were >50%
and radiochemical purities were >95%.
[0222] Competition Binding Assay
[0223] The binding affinities of nonlabelled peptides for CXCR4
were determined using a competition binding assay using the
CHO:CXCR4 cells. Briefly, CHO:CXCR4 cells (200,000 cells/well) were
plated in a 24-well BioCoat.TM. Poly-D-Lysine Multiwell Plates
(Corning) the previous night. The next day, each well was incubated
with RPMI-1640 medium (Life Technologies Corporations) supplemented
with 20 mM HEPES and 2 mg/mL BSA, [.sup.125I]SDF-1.alpha. (0.01 nM,
Perkin Elmer) and competing non-radioactive ligands (10 .mu.M to 1
.mu.M) and incubated for 1-1.5 hours at 27.degree. C. with moderate
shaking. After incubation, the cells were washed with ice-cold PBS
twice, trypsinized and counted on a Perkin Elmer WIZARD 2480 gamma
counter. IC50 values were determined by a nonlinear regression
analysis to fit a logistic equation to the competition data using
GraphPad Prism 7.
[0224] Internalization
[0225] 2.times.10.sup.6 cells/well are seeded in complete growth
medium in a 24 well Poly-D-Lysine plate (Corning BioCoat.TM., Ref
No. 354414) 24-48 hours prior to the assay. Reactions are performed
in triplicate, an unblocked and a blocked set for both CHOwt and
CHO::CXCR4 cells. For the assay, the growth medium is replaced with
400 .mu.l of reaction medium (RPMI, 2 mg/ml BSA, 20 mM HEPES). For
blocked sets, cells are preincubated for 1 hour with 1 .mu.M
LY2510924 at 37.degree. C. and 5% CO.sub.2. 0.8 MBq of 68Ga-BL02
per well is added to both unblocked and blocked wells and incubated
at 27.degree. C. with mild shaking for 1 hour. 3 samples of the
radiolabelled peptide with no cells will be used as standard. The
supernatant is removed, and cells are washed once with ice-cold
PBS. The cells were then washed twice with washes with 200 .mu.L of
ice-cold 0.2 M Acetic Acid, 0.5 M NaCl, pH2.6. The washings were
combined and measured, constituting the membrane bound portion of
peptide. The cells are washed again with ice-cold PBS, trypsinized,
collected and measured, constituting the internalized fraction of
the peptide. Standards, membrane bound fraction and cells are
counted on the Wizard gamma counter. Analysis is performed using
GraphPad Prism.
[0226] Cell Culture
[0227] The Daudi B lymphoblast cell line (ATCC.RTM. CCL-213) and
PC-3 prostate adenocarcinoma (ATCC.RTM. CRL-1435) were purchased
from the American Type Culture Collection and tested for potential
rodent pathogens and mycoplasma contamination using the IMPACT test
(IDEXX BioAnalytics). The CHO:CXCR4 cell line was a kind gift from
Drs. David McDermott and Xiaoyuan Chen (National Institutes of
Health). The GRANTA519, Jeko1 and Z138 cells were a kind gift from
Dr. Christian Steidl. The Daudi, GRANTA519, Jeko1, Z138, PC-3 and
CHO:CXCR4 cells were cultured in a 5% CO.sub.2 atmosphere at
37.degree. C. in a humidified incubator.
[0228] The Daudi and GRANTA519 cells were cultured with RPMI-1640
medium (Life Technologies Corporations) supplemented with 10% fetal
bovine serum (Sigma-Aldrich), 100 I.U./mL penicillin, and 100
.mu.g/mL streptomycin (Penicillin-Streptomycin Solution). The Jeko1
cells were cultured with RPMI-1640 medium (Life Technologies
Corporations) supplemented with 20% fetal bovine serum
(Sigma-Aldrich), 100 I.U./mL penicillin, and 100 .mu.g/mL
streptomycin (Penicillin-Streptomycin Solution). The Z138 cells
were cultured with Iscove's Modified Dulbecco's Medium (IMDM)
supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100
I.U./mL penicillin, and 100 .mu.g/mL streptomycin
(Penicillin-Streptomycin Solution). The CHO:CXCR4 cells and PC-3
cells were cultured with F12K medium (Life Technologies
Corporations) supplemented with 10% fetal bovine serum
(Sigma-Aldrich), 100 I.U./mL penicillin, and 100 .mu.g/mL
streptomycin (Penicillin-Streptomycin Solution).
[0229] Animal Models
[0230] Animal experiments were performed in accordance with
guidelines established by the Canadian Council on Animal Care,
under a research protocol approved by the Animal Ethics Committee
of the University of British Columbia. For Daudi, Z138, GRANTA519
and Jeko1 xenografts, male
NOD.Cg-Rag1.sup.tm1MomII2rg.sup.tm1Wjl/SzJ (NRG) mice were
subcutaneously inoculated on the left flank with 5.times.10.sup.6
cells (100 .mu.L; 1:1 ratio of PBS/Matrigel) and tumors were grown
to a size of 200-500 mm.sup.3. For PC-3 xenografts, male
NOD.Cg-Rag1.sup.tm1MomII2rg.sup.tm1Wjl/SzJ (NRG) mice were
subcutaneously inoculated on the left flank with 5.times.10.sup.6
PC-3 cells (100 .mu.L; 1:1 ratio of PBS/Matrigel) and tumors were
grown to a size of 200-400 mm.sup.3.
[0231] PET/CT Imaging
[0232] PET and CT scans were performed on a Siemens Inveon
microPET/CT with body temperature maintained by a heating pad.
Tumor-bearing mice were briefly sedated with isoflurane (2-2.5%
isoflurane in 2 L/min 02) for i.v. injection of 4-7 MBq of each PET
radiotracer. As a blocking control, mice received intraperitoneal
(i.p.) injection of 7.5 .mu.g LY2510924 15 minutes prior to
radiotracer administration. The animals were allowed to roam freely
during the uptake period (50 or 110 minutes), after which they were
sedated and scanned. The CT scan was obtained for attenuation
correction and anatomical localization (80 kV; 500 pA; 3 bed
positions; 34% overlap; 220.degree. continuous rotation) followed
by a 10 min PET acquisition at 1 or 2 h p.i. of the radiotracer.
PET data were acquired in list mode, reconstructed using
3-dimensional ordered-subsets expectation maximization (2
iterations) followed by a fast maximum a priori algorithm (18
iterations) with CT-based attenuation correction. Images were
analyzed using the Inveon Research Workplace software (Siemens
Healthineers).
[0233] Biodistribution
[0234] Under isoflurane anesthesia (2-2.5% isoflurane in 2 L/min
O.sub.2), the mice were injected intravenously with 0.8-3.0 MBq of
each radiotracer. Additional groups of mice received 7.5 .mu.g
LY2510924 as a blocking control i.p. 15 min before radiotracer
injection. The mice were euthanized via CO.sub.2 inhalation while
anesthetized with isoflurane. Tissues were harvested, washed in
PBS, blotted dry, weighed, and measured on a Hidex AMG Automatic
Gamma Counter. The radioactivity counts were decay corrected,
converted to absolute units using a calibration curve, and
expressed as the percent injected dose per gram of tissue (%
ID/g).
[0235] In Vivo Stability
[0236] Radiolabeled peptides (10-30 MBq) was intravenously injected
into male NRG mice. After a 5-min, 24-hour or 120-hour uptake
period, mice were sedated/euthanized, and blood was collected. The
plasma was isolated and analyzed with analytical radio-HPLC
following published procedures (Lin et al., Cancer Res. 2015,
75:387-393).
[0237] Dosimetry
[0238] The multi-time-point organ uptake obtained from
biodistribution data of .sup.177Lu-labeled analogs was decayed to
their appropriate time-point, fitted to a mono-exponential or
bi-exponential model (fit chosen based on R2 and residuals) in
Python (version 3.7). The area under the curve was used to compute
residence times which were multiplied by model organ mass for human
(NURBS model) and mouse (25 g MOBY mouse phantom) for use in
OLINDA/EXM software (Hermes Medical Solution; version 2.0) that
calculated dosimetry for an average mouse (Stabin et al., J Nucl
Med. 2005, 46:1023-1027; Keenan et al., J Nucl Med. 2010,
51:471-476) and extrapolated to an average human male (Segars et
al., J Nucl Med. 2001, 42:7; Stabin et al., J Nucl Med. 2012,
53:1807-13).
[0239] Results
[0240] As shown in Tables 1-23 and FIGS. 1-11, an anionic linker
increases internalization (longer/sustained retention in tumor)
and/or facilitates background clearance. This enhances
tumor-to-background contrast for improved imaging and therapeutic
agents compared to compounds that have a cationic linker, or a
neutral linker (i.e. lysine amide conjugation or simple maleimide
conjugation). This enhances tumor-to-background contrast for
improved imaging and therapeutic agents. The albumin binder extends
the circulatory half-life of the compounds in the mouse models,
allowing for sustained uptake of the radiotracer into the tumor. As
shown in Table 24-33, the Lu-177 labeled compounds delivered high
radiation dose to tumor xenografts but minimal radiation dose to
normal tissues/organs, leading to excellent tumor-to-normal
tissue/organ therapeutic indexes. The in vivo stability of various
compounds is shown in Table 34.
TABLE-US-00005 TABLE 1 List of the binding affinities in half
maximal inhibitory constants (IC.sub.50) in the competitive binding
assay for CXCR4 of select compounds Compound IC.sub.50 (nM) n
Ga-BL02 27.9 .+-. 12.5 3 BL04 63.0 .+-. 9.0 3 BL08 11.62 .+-. 7.0 4
BL09 13.4 .+-. 2.3 4 Ga-BL17 13.0 .+-. 8.6 3 Lu-BL18 29.4 .+-. 8.7
4 Lu-BL19 17.24 .+-. 8.5 4 Ga-BL25 21.3 .+-. 0.1 2
TABLE-US-00006 TABLE 2 The internalization of [.sup.68Ga]Ga-BL02 in
CHO:CXCR4 and CHO:WT cells Cell Line Blocking % Internalized n
CHO:CXCR4 No 52.4 .+-. 1.2 3 CHO:CXCR4 Yes 11.6 .+-. 8.8 3 CHO:WT
No 13.3 .+-. 6.4 3 CHO:WT Yes 9.2 .+-. 7.3 3
TABLE-US-00007 TABLE 3 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL02 in Daudi tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked 2 h [.sup.68Ga]Ga-BL02 Mean S.Dev n Mean S.Dev n Mean
S.Dev n Blood 0.31 0.09 6 0.50 0.17 8 0.08 0.04 9 Fat 0.05 0.03 6
0.07 0.04 8 0.02 0.01 9 Testes 0.11 0.01 5 0.15 0.05 8 0.05 0.01 9
Intestine 0.22 0.03 5 0.31 0.07 8 0.12 0.04 9 Stomach 0.07 0.03 6
0.07 0.03 8 0.07 0.05 9 Spleen 0.53 0.22 6 0.23 0.07 8 0.24 0.10 9
Liver 0.59 0.07 6 0.55 0.09 8 0.54 0.04 8 Pancreas 0.18 0.14 5 0.12
0.05 8 0.05 0.02 9 Adrenals 0.36 0.10 5 0.32 0.19 8 0.21 0.07 9
Kidney 4.18 0.68 6 4.63 1.05 8 3.40 0.51 9 Lung 0.48 0.05 6 0.58
0.16 8 0.27 0.09 9 Heart 0.13 0.02 6 0.14 0.03 7 0.05 0.01 9 Muscle
0.10 0.03 5 0.09 0.03 7 0.04 0.01 9 Bone 0.19 0.06 6 0.20 0.07 8
0.09 0.03 9 Brain 0.02 0.00 6 0.02 0.01 8 0.01 0.01 9 Daudi 8.52
2.70 6 0.80 0.21 8 7.78 1.44 9 Ratios Tumor: Blood 30.57 15.01 6
1.71 0.60 8 114.6 44.60 9 Tumor: Liver 14.30 3.37 6 1.47 0.34 8
14.47 2.52 9 Tumor: Spleen 17.18 6.54 6 3.57 0.72 8 36.06 14.92 9
Tumor: Muscle 80.77 25.39 5 9.23 4.05 7 216.0 57.47 9 Tumor: Bone
48.04 21.87 6 4.44 1.62 8 87.81 21.69 9 Tumor: Lung 18.07 5.77 6
1.43 0.39 8 31.68 11.48 9
TABLE-US-00008 TABLE 4 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL02 in Z138 tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked [.sup.68Ga]Ga-BL02 Mean S.Dev n Mean S.Dev n Blood 0.30
0.03 5 0.83 0.37 6 Fat 0.04 0.01 4 0.14 0.07 6 Testes 0.11 0.02 5
0.26 0.12 5 Intestine 0.19 0.04 5 0.61 0.27 6 Stomach 0.07 0.05 4
0.16 0.09 6 Spleen 0.33 0.05 5 0.43 0.16 6 Liver 0.48 0.04 5 0.55
0.14 6 Pancreas 0.08 0.01 5 0.21 0.09 6 Adrenals 0.14 0.04 5 0.59
0.61 6 Kidney 3.48 0.29 5 7.86 4.68 6 Lung 0.50 0.08 5 0.85 0.33 6
Heart 0.11 0.01 5 0.24 0.10 6 Muscle 0.05 0.01 5 0.15 0.08 6 Bone
0.08 0.02 5 0.20 0.12 6 Brain 0.01 0.01 5 0.02 0.01 6 Z138 12.94
1.28 5 2.86 0.94 6 Ratios Tumor: Blood 43.12 2.57 5 3.94 1.58 6
Tumor: Liver 26.89 0.69 5 5.23 1.14 6 Tumor: Spleen 39.22 5.34 5
7.40 2.71 6 Tumor: Muscle 240.96 32.87 5 21.59 8.59 6 Tumor: Bone
163.06 54.42 5 19.08 11.43 6 Tumor: Lung 26.39 3.20 5 3.65 1.20
6
TABLE-US-00009 TABLE 5 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL02 in Jeko1 tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked [.sup.68Ga]Ga-BL02 Mean S.Dev n Mean S.Dev n Blood 0.38
0.10 7 1.08 0.67 6 Fat 0.05 0.02 7 0.14 0.06 6 Testes 0.14 0.06 7
0.29 0.11 6 Intestine 0.19 0.03 7 0.44 0.14 6 Stomach 0.06 0.03 7
0.13 0.07 6 Spleen 0.70 0.33 7 0.57 0.36 6 Liver 0.77 0.16 7 0.55
0.13 6 Pancreas 0.09 0.02 7 0.26 0.12 6 Adrenals 0.23 0.17 7 0.47
0.56 6 Kidney 3.32 0.41 7 8.91 6.31 6 Lung 0.74 0.09 7 1.05 0.37 6
Heart 0.13 0.02 7 0.35 0.13 6 Muscle 0.06 0.02 5 0.20 0.10 6 Bone
0.47 0.34 7 0.18 0.06 6 Brain 0.02 0.00 7 0.03 0.01 6 Jeko1 11.45
1.14 7 1.32 0.40 6 Ratios Tumor: Blood 31.30 7.38 7 1.74 1.23 6
Tumor: Liver 15.41 3.01 7 2.59 1.26 6 Tumor: Spleen 19.78 9.26 7
3.10 1.96 6 Tumor: Muscle 180.39 34.73 5 8.71 6.61 6 Tumor: Bone
37.23 22.85 7 8.83 4.89 6 Tumor: Lung 15.76 2.88 7 1.48 0.86 6
TABLE-US-00010 TABLE 6 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL02 in GRANTA519 tumor- bearing mice at selected
time points. Mice in the 1 h blocked group received an injection of
7.5 .mu.g of LY2510924 (i.p.) 15 min before tracer administration.
1 h 1 h blocked [.sup.68Ga]Ga-BL021 Mean S. Dev n Mean S. Dev n
Blood 0.41 0.11 6 0.83 0.34 6 Fat 0.05 0.02 5 0.10 0.05 6 Testes
0.16 0.04 6 0.20 0.04 6 Intestine 0.22 0.04 6 0.46 0.16 6 Stomach
0.05 0.02 5 0.12 0.05 5 Spleen 0.49 0.05 6 0.53 0.11 6 Liver 0.09
0.03 6 0.19 0.09 6 Pancreas 0.20 0.08 6 0.34 0.16 6 Adrenals 3.48
0.47 6 7.40 4.53 6 Kidney 0.62 0.11 6 0.81 0.30 6 Lung 0.12 0.01 5
0.25 0.11 6 Heart 0.49 0.05 5 0.53 0.11 6 Muscle 0.07 0.02 6 0.14
0.05 6 Bone 0.16 0.12 6 0.25 0.20 6 Brain 0.01 0.00 6 0.02 0.01 6
GRANTA519 5.50 0.95 6 0.72 0.18 6 Ratios Tumor: Blood 13.83 1.47 6
0.94 0.30 6 Tumor: Liver 11.28 1.73 6 1.37 0.21 6 Tumor: Spleen
15.86 1.94 6 1.78 0.26 6 Tumor: Muscle 83.17 15.89 6 5.36 1.51 6
Tumor: Bone 45.39 20.98 6 4.27 2.51 6 Tumor: Lung 9.12 0.55 5 0.96
0.31 6
TABLE-US-00011 TABLE 7 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL02 in PC3 tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked 2h [.sup.68Ga]Ga-BL02 Mean S.Dev n Mean S.Dev n Mean
S.Dev n Blood 0.33 0.06 3 0.54 0.18 2 0.06 0.02 3 Fat 0.20 0.25 3
0.16 0.02 2 0.01 0.00 3 Testes 0.18 0.05 3 0.35 0.15 2 0.06 0.02 3
Intestine 0.18 0.05 3 0.25 0.10 2 0.14 0.08 3 Stomach 0.05 0.02 3
0.06 0.02 2 0.06 0.02 3 Spleen 0.28 0.01 3 0.37 0.06 2 0.25 0.04 3
Liver 0.55 0.04 3 0.50 0.13 2 0.73 0.28 3 Pancreas 0.10 0.02 3 0.12
0.04 2 0.04 0.01 3 Adrenals 0.18 0.02 3 0.18 0.01 2 0.10 0.02 3
Kidney 3.60 0.36 3 3.75 1.72 2 4.67 2.19 3 Lung 0.50 0.03 3 0.60
0.15 2 0.33 0.11 3 Heart 0.14 0.02 3 0.21 0.06 2 0.06 0.02 3 Muscle
0.08 0.03 3 0.11 0.03 2 0.03 0.01 3 Bone 0.12 0.03 3 0.15 0.07 2
0.11 0.05 3 Brain 0.01 0.00 3 0.02 0.00 2 0.01 0.00 3 PC3 1.83 0.44
3 0.75 0.26 2 1.41 0.37 3 Ratios Tumor: Blood 5.74 1.67 3 1.56 1.01
2 25.33 3.89 3 Tumor: Liver 3.31 0.74 3 1.62 0.94 2 1.99 0.25 3
Tumor: Spleen 6.62 1.69 3 2.16 1.10 2 5.63 0.61 3 Tumor: Muscle
23.74 9.95 3 7.68 4.58 2 55.01 0.87 3 Tumor: Bone 15.68 5.15 3 6.16
4.62 2 13.96 3.28 3 Tumor: Lung 3.71 1.15 3 1.36 0.77 2 4.36 0.39
3
TABLE-US-00012 TABLE 8 Biodistribution data (% ID/g) of
[.sup.18F]F-BL04 in Daudi tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked 2 h [.sup.18F]F-BL04 Mean S. Dev n Mean S.Dev n Mean
S.Dev n Blood 0.34 0.06 7 0.44 0.15 7 0.08 0.02 7 Fat 0.05 0.03 6
0.06 0.03 7 0.02 0.02 7 Testes 0.21 0.15 7 0.13 0.04 7 0.04 0.01 7
Intestine 0.20 0.02 7 0.25 0.04 7 0.11 0.03 7 Stomach 0.06 0.05 7
0.06 0.04 7 0.03 0.01 7 Spleen 0.27 0.34 7 0.17 0.08 7 0.10 0.03 7
Liver 0.20 0.03 7 0.21 0.03 7 0.15 0.02 7 Pancreas 0.10 0.03 7 0.11
0.04 7 0.04 0.01 7 Adrenals 0.27 0.26 7 0.26 0.11 7 0.22 0.20 7
Kidney 18.39 2.43 7 21.72 4.14 7 15.30 2.30 7 Lung 0.35 0.06 7 0.40
0.14 7 0.20 0.04 7 Heart 0.11 0.03 7 0.13 0.04 7 0.05 0.01 7 Muscle
0.07 0.01 7 0.10 0.06 7 0.04 0.03 7 Bone 0.21 0.02 7 0.22 0.06 7
0.18 0.05 7 Brain 0.01 0.00 7 0.01 0.00 7 0.01 0.00 7 Daudi 3.08
0.59 7 0.47 0.23 7 2.16 0.89 7 Ratios Tumor Blood 7.92 3.63 7 1.20
0.72 7 28.97 14.55 7 Tumor:Liver 15.64 1.97 7 2.29 1.07 7 14.67
5.66 7 Tumor:Spleen 19.99 8.97 7 2.87 1.26 7 23.89 11.64 7
Tumor:Muscle 45.63 7.15 7 5.81 3.63 7 61.14 27.56 7 Tumor:Bone
14.84 3.45 7 2.44 1.47 7 12.77 6.25 7 Tumor:Lungs 8.93 1.26 7 1.31
0.76 7 10.75 3.76 7
TABLE-US-00013 TABLE 9 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL06 in Daudi tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked 2 h [.sup.68Ga]Ga-BL06 Mean S.Dev n Mean S.Dev n Mean
S.Dev n Blood 2.78 0.46 6 1.17 0.26 7 1.11 0.24 8 Fat 0.35 0.10 6
0.16 0.09 7 0.19 0.04 8 Testes 0.61 0.10 6 0.34 0.06 7 0.46 0.02 8
Intestine 1.07 0.13 6 0.70 0.18 7 0.68 0.11 8 Stomach 0.29 0.08 6
0.14 0.04 7 0.21 0.07 8 Spleen 15.53 1.83 6 3.30 0.73 7 9.14 1.60 8
Liver 8.60 0.77 6 9.25 0.94 6 10.35 0.40 8 Pancreas 0.70 0.09 6
0.29 0.08 7 0.39 0.07 7 Adrenals 3.61 1.14 5 0.80 0.33 7 3.19 1.11
8 Kidney 6.25 1.05 6 8.26 2.79 7 4.82 0.61 8 Lung 15.00 2.06 6 2.20
0.55 7 7.18 0.86 8 Heart 1.73 0.25 6 0.48 0.11 7 0.93 0.17 8 Muscle
0.41 0.11 6 0.19 0.04 7 0.21 0.06 8 Bone 1.84 0.65 6 0.82 0.26 7
1.11 0.25 8 Brain 0.06 0.00 6 0.03 0.01 7 0.03 0.00 8 Daudi 10.26
1.29 6 2.06 0.62 7 11.32 1.44 8 Ratios Tumor:Blood 3.75 1.18 6 1.84
0.74 7 10.35 1.33 8 Tumor:Liver 1.19 0.56 6 0.21 0.05 6 1.10 0.14 8
Tumor:Spleen 0.67 0.17 6 0.64 0.19 7 1.25 0.13 8 Tumor:Muscle 25.59
4.15 6 10.97 2.86 7 57.98 13.51 8 Tumor:Bone 6.04 1.30 6 2.77 1.10
7 10.56 2.36 8 Tumor:Lungs 0.69 0.21 6 0.95 0.26 7 1.59 0.21 8
TABLE-US-00014 TABLE 10 Ex vivo biodistribution data of
[.sup.18F]F-BL08 (% ID/g) in Daudi xenograft bearing mice. The mice
in the 1 h blocked group received an intraperitoneal injection of
7.5 .mu.g of LY2510924 15 minutes prior to radiotracer
administration. 1 h 1 h blocked 2 h [.sup.18F]F-BL08 Mean S.Dev n
Mean S. Dev n Mean S. Dev n Blood 0.36 0.05 6 0.83 0.46 8 0.09 0.05
7 Fat 0.04 0.01 6 0.13 0.08 8 0.01 0.00 5 Testes 0.14 0.02 6 0.67
1.30 8 0.04 0.02 7 Intestine 0.19 0.03 6 0.32 0.19 7 0.09 0.03 7
Stomach 0.053 0.052 6 0.12 0.05 8 0.02 0.01 7 Spleen 0.32 0.17 7
0.72 0.28 8 0.13 0.03 7 Liver 0.62 0.02 6 0.18 0.11 8 0.41 0.09 7
Pancreas 0.08 0.01 6 0.42 0.42 8 0.03 0.02 7 Adrenals 0.23 0.16 6
6.70 3.21 8 0.12 0.10 7 Kidney 3.47 0.46 6 0.86 0.48 8 2.15 0.46 7
Lung 0.61 0.17 6 0.24 0.13 8 0.27 0.12 7 Heart 0.13 0.01 6 0.72
0.28 8 0.04 0.02 7 Muscle 0.07 0.01 5 0.14 0.08 8 0.03 0.02 5 Bone
0.18 0.05 6 0.32 0.16 8 0.12 0.04 7 Brain 0.01 0.01 6 0.02 0.01 8
0.01 0.00 7 Daudi 7.60 1.38 6 1.17 0.71 8 5.67 1.25 7 Ratios
Tumour: Blood 21.53 6.49 6 2.01 2.13 8 71.90 19.76 7 Tumour:Liver
13.05 4.05 6 1.88 1.54 8 14.04 2.99 7 Tumour: Spleen 25.81 4.57 6
3.87 3.28 8 43.46 7.98 7 Tumour:Muscle 107.7 24.86 5 12.30 13.85 8
339.0 81.39 5 Tumour: Bone 45.87 17.31 6 4.49 4.18 8 52.27 12.27 7
Tumour:Lung 13.18 3.88 6 1.85 1.88 8 23.08 5.57 7
TABLE-US-00015 TABLE 11 Ex vivo biodistribution data of
[.sup.18F]F-BL09 (% ID/g) in Daudi xenograft bearing mice. The mice
in the 1 h blocked group received an intraperitoneal injection of
7.5 .mu.g of LY2510924 15 minutes prior to radiotracer
administration. 1 h 1 h blocked 2 h [.sup.18F]F-BL09 Mean S. Dev n
Mean S. Dev n Mean S. Dev n Blood 0.44 0.11 7 0.53 0.30 7 0.10 0.03
7 Fat 0.05 0.02 7 0.10 0.06 7 0.01 0.00 7 Testes 0.21 0.06 7 0.21
0.16 7 0.05 0.02 7 Intestine 0.23 0.08 7 0.26 0.12 7 0.14 0.08 6
Stomach 0.06 0.02 7 0.09 0.04 7 0.01 0.00 7 Spleen 0.31 0.08 7 0.24
0.12 7 0.12 0.03 7 Liver 0.56 0.09 7 0.50 0.20 7 0.35 0.05 6
Pancreas 0.11 0.05 6 0.14 0.08 7 0.03 0.01 6 Adrenals 0.34 0.32 7
0.17 0.09 7 0.06 0.03 7 Kidney 9.74 1.47 7 8.65 3.23 7 7.59 1.00 7
Lung 0.54 0.15 7 0.53 0.24 7 0.20 0.03 6 Heart 0.16 0.04 7 0.18
0.10 7 0.04 0.01 6 Muscle 0.08 0.03 6 0.07 0.02 5 0.03 0.01 6 Bone
0.34 0.23 7 0.29 0.28 7 0.16 0.09 7 Brain 0.02 0.00 7 0.02 0.02 7
0.01 0.00 7 Daudi 6.61 2.07 6 0.79 0.65 7 5.83 0.92 7 Ratios
Tumour: Blood 15.42 2.27 6 1.87 1.44 7 64.36 19.84 7 Tumour:Liver
11.82 2.94 6 1.58 1.05 7 17.07 3.01 6 Tumour: Spleen 21.79 5.29 6
3.82 2.87 7 49.37 10.59 7 Tumour:Muscle 83.21 19.44 5 11.15 8.52 5
238.60 71.96 6 Tumour: Bone 25.95 11.94 6 4.61 3.95 7 49.94 26.98 7
Tumour:Lung 12.59 2.04 6 1.70 1.23 7 29.99 9.45 6
TABLE-US-00016 TABLE 12 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL17 in Daudi tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked 2 h [.sup.68Ga]Ga-BL17 Mean S. Dev n Mean S. Dev n Mean
S. Dev n Blood 0.41 0.12 4 1.40 0.14 2 0.09 0.01 4 Fat 0.04 0.01 4
0.32 0.01 2 0.02 0.01 4 Testes 0.20 0.05 4 0.53 0.16 2 0.06 0.01 4
Intestine 0.21 0.07 4 0.65 0.08 2 0.15 0.07 4 Stomach 0.05 0.03 4
0.18 0.02 2 0.03 0.02 4 Spleen 0.33 0.07 4 0.81 0.20 2 0.30 0.05 4
Liver 0.50 0.07 4 0.84 0.13 2 0.47 0.08 4 Pancreas 0.10 0.03 4 0.38
0.05 2 0.04 0.02 4 Adrenals 0.16 0.07 4 0.79 0.25 2 0.20 0.12 4
Kidney 3.30 0.41 4 15.17 8.21 2 3.28 0.34 4 Lung 0.51 0.12 4 1.35
0.20 2 0.25 0.03 4 Heart 0.14 0.04 4 0.45 0.05 2 0.06 0.01 4 Muscle
0.10 0.03 4 0.25 0.00 2 0.03 0.01 4 Bone 0.09 0.03 4 0.31 0.05 2
0.09 0.03 4 Brain 0.02 0.01 4 0.03 0.00 2 0.01 0.00 4 Daudi 6.32
0.67 4 0.68 0.00 2 6.40 1.60 4 Ratios Tumour: Blood 16.94 6.57 4
0.49 0.05 2 69.33 18.61 4 Tumour:Liver 12.66 0.79 4 0.81 0.13 2
13.69 4.46 4 Tumour: Spleen 19.76 3.57 4 0.87 0.22 2 21.11 6.42 4
Tumour:Muscle 67.93 16.71 4 2.74 0.05 2 256.87 72.49 4 Tumour: Bone
76.60 21.25 4 2.24 0.35 2 79.15 32.79 4 Tumour:Lung 12.64 2.48 4
0.51 0.07 2 25.37 6.19 4
TABLE-US-00017 TABLE 13 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL20 in Daudi tumor-bearing mice at selected time
points. Mice in the 1 h blocked group received an injection of 7.5
.mu.g of LY2510924 (i.p.) 15 min before tracer administration. 1 h
1 h blocked 2 h [.sup.68Ga]Ga-BL17 Mean S. Dev n Mean S. Dev n Mean
S. Dev n Blood 0.42 0.12 3 0.65 na 1 0.08 0.02 4 Fat 0.06 0.03 3
0.12 na 1 0.01 0.01 4 Testes 0.15 0.03 3 0.20 na 1 0.05 0.01 4
Intestine 0.21 0.02 3 0.37 na 1 0.25 0.25 4 Stomach 0.05 0.01 3
0.24 na 1 0.05 0.03 4 Spleen 0.49 0.07 3 0.30 na 1 0.21 0.06 4
Liver 0.58 0.07 3 0.44 na 1 0.51 0.04 4 Pancreas 0.11 0.03 3 0.16
na 1 0.04 0.01 4 Adrenals 0.35 0.20 3 0.35 na 1 0.27 0.17 4 Kidney
3.58 0.26 3 5.92 na 1 3.02 0.22 4 Lung 0.51 0.08 3 0.71 na 1 0.22
0.04 4 Heart 0.15 0.03 3 0.22 na 1 0.05 0.01 4 Muscle 0.08 0.02 3
0.13 na 1 0.02 0.00 4 Bone 0.17 0.00 3 0.15 na 1 0.05 0.03 4 Brain
0.01 0.00 3 0.02 na 1 0.01 0.00 4 Daudi 9.07 0.76 3 0.59 na 1 8.01
1.39 4 Ratios Tumour: Blood 22.78 7.35 3 0.90 na 1 113.17 45.03 4
Tumour: Liver 15.68 0.90 3 1.34 na 1 15.57 1.73 4 Tumour:Spleen
18.55 1.38 3 1.96 na 1 41.58 13.90 4 Tumour:Muscle 125.40 54.59 3
4.49 na 1 346.93 81.84 4 Tumour: Bone 54.57 6.00 3 3.88 na 1 189.39
109.18 4 Tumour:Lung 18.12 3.56 3 0.82 na 1 37.40 8.71 4
TABLE-US-00018 TABLE 14 Biodistribution data (%|D/g) of
[.sup.68Ga]Ga-BL25 in Daudi tumor-bearing mice at selected time
points. 2 h [.sup.68Ga]Ga-BL25 Mean S.Dev n Blood 0.18 0.01 4 Fat
0.02 0.01 4 Testes 0.07 0.00 4 Intestine 0.13 0.03 4 Stomach 0.03
0.02 4 Spleen 0.19 0.03 4 Liver 0.47 0.04 4 Pancreas 0.05 0.01 4
Adrenals 0.10 0.01 4 Kidney 1.92 0.07 4 Lung 0.37 0.11 4 Heart 0.07
0.00 4 Muscle 0.04 0.00 4 Bone 0.06 0.02 4 Brain 0.01 0.00 4 Daudi
5.53 0.29 4 Ratios Tumor:Blood 30.77 1.18 4 Tumor:Liver 12.05 0.76
4 Tumor:Spleen 30.72 3.73 4 Tumor:Muscle 159.64 19.97 4 Tumor:Bone
103.83 33.98 4 Tumor:Lung 16.13 4.21 4
TABLE-US-00019 TABLE 15 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL22 in Daudi tumor-bearing mice at selected time
points. 1 h 3 h [.sup.68Ga]Ga-BL22 Mean S. Dev n Mean S. Dev n
Blood 14.23 0.62 4 12.89 0.70 4 Fat 0.74 0.10 4 0.81 0.09 4 Testes
1.73 0.36 4 2.26 0.30 4 Intestine 1.39 0.18 4 1.21 0.13 4 Stomach
0.51 0.15 4 0.56 0.16 4 Spleen 2.76 0.37 4 2.64 0.56 4 Liver 3.23
0.43 4 3.50 0.29 4 Pancreas 1.38 0.08 4 1.36 0.12 4 Adrenals 2.96
0.42 4 3.04 0.61 4 Kidney 5.18 0.16 4 4.90 0.29 4 Lung 9.83 0.85 4
7.51 0.84 4 Heart 2.81 0.16 4 2.89 0.09 4 Muscle 1.08 0.18 4 0.98
0.02 4 Bone 0.96 0.23 4 1.16 0.15 4 Brain 0.19 0.02 4 0.18 0.01 4
Daudi 3.93 0.67 4 9.04 0.41 4 Ratios Tumor:Blood 0.28 0.05 4 0.70
0.05 4 Tumor:Liver 1.26 0.21 4 2.60 0.34 4 Tumor:Spleen 1.49 0.38 4
3.54 0.73 4 Tumor:Muscle 3.84 1.04 4 9.23 0.31 4 Tumor:Bone 4.42
1.54 4 7.90 1.41 4 Tumor:Lung 0.41 0.04 4 1.21 0.11 4
TABLE-US-00020 TABLE 16 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL23 in Daudi tumor-bearing mice at selected time
points. 1 h 3 h [.sup.68Ga]Ga-BL23 Mean S. Dev n Mean S. Dev n
Blood 9.93 0.27 4 4.48 0.45 4 Fat 0.55 0.08 4 0.28 0.02 4 Testes
1.29 0.08 4 1.08 0.35 4 Intestine 0.96 0.12 4 0.53 0.01 4 Stomach
0.31 0.06 4 0.18 0.02 4 Spleen 2.42 0.36 4 1.43 0.08 4 Liver 2.83
0.17 4 1.84 0.09 4 Pancreas 1.13 0.09 4 0.58 0.03 4 Adrenals 2.40
0.47 4 1.22 0.36 4 Kidney 6.08 0.22 4 4.97 0.38 4 Lung 5.82 1.23 4
2.93 0.49 4 Heart 2.37 0.11 4 1.08 0.03 4 Muscle 0.73 0.07 4 0.39
0.03 4 Bone 0.95 0.17 4 0.47 0.05 4 Brain 0.14 0.01 4 0.08 0.01 4
Daudi 7.59 1.17 4 10.88 0.80 4 Ratios Tumor:Blood 0.84 0.17 4 2.46
0.42 4 Tumor:Liver 2.96 0.73 4 5.92 0.40 4 Tumor:Spleen 3.45 0.73 4
7.63 0.94 4 Tumor:Muscle 11.38 2.40 4 27.82 3.88 4 Tumor:Bone 8.81
1.98 4 23.23 4.02 4 Tumor:Lung 1.49 0.52 4 3.79 0.68 4
TABLE-US-00021 TABLE 17 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL27 in Daudi tumor-bearing mice at selected time
points. 1 h 3 h [.sup.68Ga]Ga-BL27 Mean S. Dev n Mean S. Dev n
Blood 4.60 0.50 4 0.95 0.12 4 Fat 0.35 0.04 4 0.09 0.03 4 Testes
0.80 0.11 4 0.31 0.03 4 Intestine 0.85 0.11 4 0.36 0.07 4 Stomach
0.19 0.07 4 0.12 0.05 4 Spleen 2.82 0.64 4 1.06 0.20 4 Liver 2.61
0.04 4 2.64 0.33 4 Pancreas 0.62 0.09 4 0.26 0.12 4 Adrenals 1.66
0.32 4 0.58 0.04 4 Kidney 6.21 0.49 4 4.77 0.43 4 Lung 7.23 0.88 4
2.10 0.61 4 Heart 1.27 0.25 4 0.32 0.09 4 Muscle 0.43 0.07 4 0.13
0.04 4 Bone 0.66 0.06 4 0.27 0.02 4 Brain 0.07 0.01 4 0.03 0.01 4
Daudi 8.78 1.36 4 10.37 1.70 4 Ratios Tumor:Blood 1.99 0.44 4 10.85
0.82 4 TumonLiver 3.46 0.49 4 3.91 0.22 4 Tumor:Spleen 3.37 1.01 4
10.08 2.23 4 Tumor:Muscle 21.47 5.50 4 83.68 16.22 4 Tumor:Bone
13.68 2.10 4 38.31 6.36 4 Tumor:Lung 1.27 0.27 4 5.59 3.03 4
TABLE-US-00022 TABLE 18 Biodistribution data (% ID/g) of
[.sup.68Ga]Ga-BL28 in Daudi tumor-bearing mice at selected time
points. 1 h 3 h [.sup.68Ga]Ga-BL28 Mean S. Dev n Mean S. Dev n
Blood 14.70 1.47 4 11.59 0.33 4 Fat 0.64 0.20 4 0.56 0.09 4 Testes
1.73 0.24 4 2.05 0.24 4 Intestine 1.21 0.12 4 1.00 0.05 4 Stomach
0.47 0.07 4 0.38 0.05 4 Spleen 2.21 0.08 4 2.34 0.34 4 Liver 2.92
0.52 4 2.92 0.34 4 Pancreas 1.49 0.22 4 1.21 0.08 4 Adrenals 2.53
0.70 4 2.46 0.78 4 Kidney 5.41 0.60 4 4.76 0.42 4 Lung 7.11 1.01 4
5.35 0.19 4 Heart 3.21 0.37 4 2.29 0.14 4 Muscle 0.96 0.14 4 0.76
0.09 4 Bone 1.14 0.25 4 0.94 0.15 4 Brain 0.19 0.02 4 0.17 0.02 4
Daudi 4.59 1.16 4 7.82 0.51 4 Ratios Tumor:Blood 0.31 0.04 4 0.68
0.05 4 Tumor:Liver 1.59 0.30 4 2.71 0.44 4 Tumor:Spleen 2.08 0.43 4
3.38 0.44 4 Tumor:Muscle 4.82 0.56 4 10.49 1.90 4 Tumor:Bone 4.12
0.81 4 8.55 1.86 4 Tumor:Lung 0.65 0.13 4 1.46 0.10 4
TABLE-US-00023 TABLE 19 Biodistribution data (% ID/g) of
[.sup.177Lu]Lu-BL02 in Z138 tumor-bearing mice at selected time
points. 1 h 1 h blocked 4 h 24 h 72 h [.sup.177Lu]Lu-BL02 Mean S.
Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Blood
0.44 0.07 6 2.06 1.96 6 0.03 0.00 7 0.010 0.001 7 0.007 0.003 5 Fat
0.07 0.03 6 0.19 0.06 6 0.03 0.06 7 0.011 0.002 7 0.008 0.003 6
Testes 0.18 0.02 6 0.39 0.15 6 0.04 0.00 6 0.032 0.013 7 0.029
0.006 6 Intestine 0.27 0.05 6 0.75 0.31 6 0.09 0.02 6 0.126 0.184 7
0.023 0.003 6 Stomach 0.12 0.07 6 0.20 0.09 6 0.07 0.07 7 0.052
0.028 7 0.022 0.007 6 Spleen 0.56 0.20 6 0.56 0.15 6 0.33 0.08 7
0.277 0.036 7 0.288 0.122 6 Liver 0.93 0.14 6 0.99 0.25 6 0.92 0.09
7 0.703 0.030 7 0.436 0.073 6 Pancreas 0.14 0.02 6 0.31 0.10 6 0.04
0.01 7 0.034 0.003 7 0.016 0.002 6 Adrenals 0.46 0.29 6 0.47 0.30 6
0.13 0.10 7 0.117 0.072 7 0.073 0.024 6 Kidney 3.87 0.66 6 10.90
6.47 6 3.27 0.40 7 1.433 0.204 7 0.560 0.078 6 Lung 0.83 0.19 6
1.57 0.89 6 0.26 0.04 7 0.176 0.046 7 0.184 0.091 6 Heart 0.18 0.03
6 0.46 0.24 6 0.05 0.01 7 0.035 0.005 7 0.022 0.003 6 Muscle 0.10
0.02 6 0.45 0.48 6 0.03 0.01 7 0.019 0.004 7 0.010 0.002 6 Bone
0.25 0.11 6 0.49 0.32 6 0.11 0.02 7 0.085 0.027 7 0.053 0.010 6
Brain 0.02 0.00 6 0.04 0.01 6 0.01 0.00 7 0.003 0.001 7 0.001 0.000
6 Z138 17.17 3.04 6 3.75 0.83 6 15.57 2.59 7 8.791 1.215 6 3.574
0.645 6 Ratios Tumour: Blood 38.98 3.01 6 2.74 1.22 6 627.62 129.95
7 873.49 84.55 7 613.24 330.77 5 Tumour: Liver 18.53 2.34 6 3.84
0.40 6 16.93 1.54 7 12.51 1.72 7 8.32 1.60 6 Tumour:Spleen 33.05
8.73 6 6.87 1.25 6 49.43 10.83 7 31.90 3.61 7 13.86 5.15 6
Tumour:Muscle 166.59 14.05 6 13.22 5.59 6 503.09 126.87 7 463.22
90.14 7 358.55 59.03 6 Tumour:Bone 74.82 20.30 6 9.53 3.81 6 145.11
15.72 7 109.48 29.98 7 68.39 12.84 6 Tumour:Lung 21.08 2.76 6 2.75
0.89 6 60.62 13.42 7 52.72 16.22 7 21.59 5.59 6
TABLE-US-00024 TABLE 20 Biodistribution data (% ID/g) of
[.sup.177Lu]Lu-BL02 in GRANTA519 tumor-bearing mice at selected
time points. 1 h 1 h blocked 4 h 24 h 72 h [.sup.177Lu]Lu-BL02 Mean
S. Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n
Blood 0.50 0.13 6 1.14 0.45 6 0.02 0.00 6 0.006 0.001 5 0.002 0.000
5 Fat 0.08 0.03 6 0.16 0.08 6 0.01 0.00 7 0.008 0.002 7 0.006 0.001
5 Testes 0.21 0.06 6 0.32 0.11 6 0.04 0.01 7 0.034 0.005 7 0.034
0.020 5 Intestine 0.28 0.02 6 0.58 0.23 6 0.09 0.04 7 0.046 0.011 5
0.017 0.005 6 Stomach 0.10 0.03 6 0.19 0.09 6 0.05 0.02 7 0.206
0.297 7 0.021 0.010 5 Spleen 0.49 0.10 6 0.48 0.17 6 0.28 0.07 7
0.321 0.139 7 0.198 0.008 6 Liver 0.96 0.15 6 1.07 0.29 6 0.87 0.16
7 0.635 0.061 7 0.413 0.047 6 Pancreas 0.17 0.05 6 0.30 0.11 6 0.04
0.00 7 0.028 0.008 7 0.015 0.003 6 Adrenals 0.39 0.23 6 0.48 0.23 6
0.10 0.03 6 0.094 0.065 7 0.346 0.517 6 Kidney 3.91 0.46 6 8.63
4.34 6 2.98 0.42 7 1.329 0.162 7 0.573 0.095 6 Lung 0.77 0.19 6
1.16 0.44 6 0.27 0.08 7 0.117 0.036 7 0.060 0.028 6 Heart 0.19 0.05
6 0.40 0.16 6 0.05 0.02 7 0.034 0.006 7 0.022 0.003 6 Muscle 0.13
0.05 6 0.34 0.21 6 0.04 0.01 7 0.015 0.003 7 0.007 0.001 6 Bone
0.24 0.04 6 0.41 0.18 6 0.09 0.01 7 0.090 0.011 7 0.061 0.007 6
Brain 0.02 0.01 6 0.04 0.02 6 0.01 0.00 7 0.003 0.001 7 0.001 0.001
6 GRANTA519 6.83 1.26 6 0.84 0.31 6 3.22 0.49 7 1.090 0.125 7 0.353
0.025 6 Ratios Tumour:Blood 14.18 2.52 6 0.77 0.17 6 119.44 45.07 6
206.07 47.16 5 126.69 44.62 5 Tumour:Liver 7.10 0.96 6 0.78 0.14 6
3.75 0.43 7 1.72 0.19 7 0.86 0.08 6 Tumour:Spleen 14.20 2.50 6 1.77
0.17 6 11.98 2.92 7 3.78 1.04 7 1.53 0.54 5 Tumour:Muscle 56.86
20.00 6 3.18 1.51 6 90.57 26.90 7 76.90 17.73 7 50.64 6.85 6
Tumour:Bone 28.95 2.63 6 2.16 0.54 6 34.26 4.03 7 12.33 2.00 7 5.88
0.57 6 Tumour:Lung 9.08 1.96 6 0.74 0.09 6 12.56 2.64 7 9.91 2.64 7
6.60 1.87 6
TABLE-US-00025 TABLE 21 Biodistribution data (% ID/g) of
[.sup.177Lu]Lu-BL18 in Daudi tumor-bearingmice at selected time
points. 1 h 4 h 24 h 72 h 120 h [.sup.177Lu]Lu-BL18 Mean S. Dev n
Mean S. Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Blood 30.38
4.39 4 18.25 1.37 4 13.49 1.15 4 6.12 0.56 4 3.35 0.76 4 Fat 1.18
0.12 4 1.30 0.23 4 0.73 0.14 4 0.63 0.23 4 0.43 0.16 4 Testes 4.02
0.75 4 3.96 0.36 4 4.28 0.53 4 4.45 0.81 4 3.88 0.61 4 Intestine
2.76 0.53 4 1.53 0.20 4 1.55 0.14 4 0.94 0.27 4 0.50 0.09 4 Stomach
0.89 0.29 4 0.96 0.36 4 1.4 10.12 4 0.73 0.29 4 0.45 0.19 4 Spleen
6.35 2.44 4 4.04 0.79 4 5.76 0.52 4 6.47 1.52 4 6.34 1.49 4 Liver
7.12 1.92 4 5.64 0.94 4 4.66 0.67 4 3.18 0.83 4 2.04 0.14 4
Pancreas 3.84 0.79 4 2.19 0.12 4 1.83 0.17 4 1.42 0.41 4 0.85 0.11
4 Adrenals 8.47 3.21 4 5.25 1.57 4 6.20 0.91 4 7.82 2.06 4 4.94
1.38 4 Kidney 8.68 2.01 4 5.98 0.23 4 4.78 0.27 4 2.99 0.79 4 1.72
0.25 4 Lung 21.59 5.79 4 11.30 1.42 4 8.94 0.34 4 5.59 1.94 4 3.0
10.49 4 Heart 7.3 11.47 4 4.88 0.70 4 3.87 0.18 4 2.6 10.59 4 1.73
0.15 4 Muscle 1.32 0.37 4 1.56 0.06 4 1.16 0.17 4 0.85 0.25 4 0.45
0.07 4 Bone 2.87 0.85 4 1.44 0.25 4 1.71 0.33 4 1.19 0.37 4 0.95
0.35 4 Brain 0.4 10.06 4 0.30 0.02 4 0.21 0.02 4 0.13 0.04 4 0.06
0.01 4 Daudi 3.49 1.04 4 5.79 0.64 4 18.6 0.45 4 23.06 2.05 3 18.3
3.64 4 Ratios Tumour:Blood 0.12 0.03 4 0.32 0.05 4 1.38 0.09 4 4.10
0.87 3 5.76 1.89 4 Tumour: Liver 0.53 0.23 4 1.05 0.24 4 4.05 0.62
4 7.88 0.64 3 8.97 1.48 4 Tumour:Spleen 0.59 0.22 4 1.49 0.40 4
3.25 0.36 4 3.90 0.41 3 2.95 0.60 4 Tumour:Muscle 2.69 0.69 4 3.72
0.54 4 16.26 2.40 4 29.58 9.82 3 41.40 7.23 4 Tumour: Bone 1.28
0.44 4 4.17 1.12 4 11.24 2.40 4 21.36 3.97 3 21.28 7.69 4 Tumour:
Lung 0.17 0.06 4 0.52 0.12 4 2.08 0.12 4 4.50 1.31 3 6.18 1.24
4
TABLE-US-00026 TABLE 22 Biodistribution data (% ID/g) of
[.sup.177Lu]Lu-BL19 in Daudi tumor-bearing mice at selected time
points. 1 h 4 h 24 h 72 h 120 h [.sup.177Lu]Lu-BL19 Mean S. Dev n
Mean S. Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Blood 29.42
2.52 4 20.08 0.59 4 12.75 1.21 4 5.66 0.30 2 3.08 0.47 2 Fat 0.77
0.10 4 1.15 0.23 4 0.85 0.43 4 0.57 0.08 2 0.42 0.05 2 Testes 3.42
0.79 4 4.02 0.43 4 4.31 1.14 4 4.49 0.49 2 3.97 0.12 2 Intestine
2.68 0.17 4 1.75 0.16 4 1.27 0.05 4 0.81 0.03 2 0.46 0.03 2 Stomach
0.64 0.15 4 0.98 0.24 4 1.14 0.15 4 0.68 0.20 2 0.33 0.04 2 Spleen
5.39 1.34 4 3.89 0.03 4 3.94 0.45 4 5.05 0.10 2 5.18 0.11 2 Liver
6.02 1.06 4 4.3 10.57 4 5.24 2.31 4 2.54 0.27 2 2.04 0.09 2
Pancreas 3.20 0.27 4 2.52 0.10 4 1.77 0.26 4 1.28 0.09 2 0.84 0.01
2 Adrenals 6.71 1.84 4 5.12 1.66 4 5.36 1.42 4 7.48 1.55 2 3.85
0.72 2 Kidney 10.59 1.05 4 6.48 0.51 4 4.65 0.79 4 2.62 0.05 2 1.78
0.03 2 Lung 17.49 3.30 4 11.48 1.18 4 8.07 1.22 4 4.87 0.12 2 2.85
0.08 2 Heart 7.43 0.65 4 4.77 0.21 4 3.79 1.29 4 2.53 0.09 2 1.76
0.10 2 Muscle 1.30 0.20 4 1.52 0.15 4 1.47 0.74 4 0.78 0.06 2 0.42
0.03 2 Bone 2.20 0.32 4 1.99 0.65 4 0.94 0.16 4 1.37 0.03 2 0.86
0.07 2 Brain 0.45 0.03 4 0.29 0.04 4 0.23 0.07 4 0.11 0.00 2 0.07
0.00 2 Daudi 2.15 0.43 4 5.44 0.43 4 15.33 2.23 4 16.63 0.14 2
12.20 1.75 2 Ratios Tumour:Blood 0.07 0.01 4 0.27 0.01 4 1.20 0.09
4 2.94 0.18 2 3.97 0.04 2 Tumour:Liver 0.37 0.12 4 1.27 0.15 4 3.36
1.54 4 6.58 0.76 2 6.01 1.13 2 Tumour:Spleen 0.42 0.13 4 1.40 0.10
4 3.92 0.65 4 3.30 0.09 2 2.35 0.29 2 Tumour:Muscle 1.67 0.25 4
3.60 0.51 4 11.63 3.32 4 21.33 1.46 2 28.67 2.03 2 Tumour:Bone 0.98
0.16 4 3.07 1.44 4 16.89 4.91 4 12.15 0.18 2 14.11 0.83 2
Tumour:Lung 0.13 0.04 4 0.48 0.06 4 1.91 0.19 4 3.42 0.06 2 4.28
0.49 2
TABLE-US-00027 TABLE 23 Biodistribution data (% ID/g) of
[.sup.177Lu]Lu-BL23 in Daudi tumor-bearing mice at selected time
points. 1 h 1 h blocked 4 h 24 h 72 h [.sup.177Lu]Lu-BL23 Mean S.
Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Mean S. Dev n Blood
13.32 0.89 4 4.98 0.30 4 0.11 0.04 4 0.03 0.01 4 0.01 0.00 4 Fat
0.68 0.08 4 0.30 0.01 4 0.06 0.01 4 0.04 0.01 4 0.02 0.01 4 Testes
2.01 0.26 4 1.42 0.23 4 0.34 0.08 4 0.05 0.01 4 0.14 0.08 4
Intestine 1.32 0.10 4 0.79 0.31 4 0.13 0.02 4 0.27 0.02 4 0.03 0.01
4 Stomach 0.43 0.08 4 0.29 0.09 4 0.17 0.03 4 0.09 0.01 4 0.02 0.00
4 Spleen 5.81 0.18 4 4.80 1.59 4 4.45 1.54 4 5.73 0.64 4 5.51 1.20
4 Liver 5.72 0.89 4 3.97 0.67 4 3.82 1.90 4 3.23 0.19 4 2.57 0.25 4
Pancreas 1.55 0.10 4 0.69 0.05 4 0.11 0.02 4 0.07 0.01 4 0.04 0.00
4 Adrenals 5.84 1.97 4 1.99 0.65 4 1.04 0.53 4 1.24 0.85 4 0.42
0.17 4 Kidney 7.23 1.23 4 5.14 0.47 4 1.86 0.47 4 0.95 0.46 4 0.40
0.05 4 Lung 17.52 2.71 4 3.82 0.22 4 0.86 0.39 4 0.53 0.29 4 0.25
0.15 4 Heart 3.22 0.36 4 1.32 0.16 4 0.21 0.05 4 0.13 0.02 4 0.07
0.00 4 Muscle 1.09 0.09 4 0.48 0.03 4 0.07 0.03 4 0.04 0.01 4 0.02
0.00 4 Bone 1.36 0.09 4 0.55 0.10 4 0.29 0.11 4 0.33 0.09 4 0.19
0.04 4 Brain 0.19 0.03 4 0.08 0.00 4 0.01 0.00 4 0.00 0.00 4 0.00
0.00 4 Daudi 8.38 0.97 4 13.24 0.85 4 11.50 0.40 4 6.50 0.08 4 3.17
0.23 4 Ratios Tumour:Blood 0.63 0.05 4 2.67 0.27 4 95.35 23.25 4
284.24 36.85 4 362.04 40.25 4 Tumour:Liver 1.51 0.39 4 3.43 0.74 4
2.55 0.60 4 2.24 0.12 4 1.24 0.12 4 Tumour:Spleen 1.45 0.20 4 3.04
1.14 4 2.79 0.94 4 1.26 0.22 4 0.60 0.14 4 Tumour:Muscle 7.65 0.41
4 27.88 3.60 4 150.51 33.19 4 182.80 41.44 4 202.59 26.09 4
Tumour:Bone 6.17 0.86 4 24.71 5.02 4 33.91 5.10 4 22.16 7.72 4
17.44 4.28 4 Tumour: Lung 0.48 0.07 4 3.47 0.23 4 11.31 2.30 4
16.32 8.16 4 15.83 7.24 4
TABLE-US-00028 TABLE 24 Absorbed Doses in mGy/MBq for the Mouse 25
g model with isotope Lu-177 based on [.sup.177Lu]Lu-BL02 in Z138
xenograft mice. Organ Lu-177 Brain 0.0246 Large Intestine 0.0691
Small Intestine 0.0767 Stomach 0.163 Heart 0.137 Kidneys 0.887
Liver 0.573 Lungs 0.363 Pancreas 0.155 Bone 0.72 Spleen 0.533
Testes 0.0615 Thyroid 0.0403 Bladder 0.0314 Remainder of the Body
0.107 Z138 Tumor 528.22
TABLE-US-00029 TABLE 25 Absorbed Doses in mGy/MBq for the Human
Extrapolated from Mouse Model with isotope Lu-177 based on
[.sup.177Lu]Lu-BL02 in Z138 xenograft mice. Organs Lu-177 Adrenals
0.00413 Brain 4.5e-05 Esophagus 0.000564 Eyes 0.000296 Gallbladder
Wall 0.000923 Left colon 0.000755 Small Intestine 0.000691 Stomach
Wall 0.000883 Right colon 0.000642 Rectum 0.000473 Heart 0.00148
Kidneys 0.0332 Liver 0.0183 Lungs 0.0126 Pancreas 0.00105 Prostate
0.000336 Salivary Glands 0.00031 Red Marrow 0.000377 Skeleton
0.000457 Spleen 0.0193 Testes 0.00168 Thymus 0.000454 Thyroid
0.000396 Urin Blad 0.00032 Remainder of the Body 0.00117
TABLE-US-00030 TABLE 26 Absorbed Doses in mGy/MBq for the Mouse 25
g model with isotope Lu-177, based on [.sup.177Lu]Lu-BL02 in
GRANTA519 xenograft mice. Organ Lu-177 Brain 0.0253 Large Intestine
0.0874 Small Intestine 0.0968 Stomach 0.137 Heart 0.133 Kidneys
0.836 Liver 0.577 Lungs 0.169 Pancreas 0.15 Bone 0.662 Spleen 0.28
Testes 0.0809 Thyroid 0.0397 Bladder 0.0331 Remainder of the Body
0.106 GRANTA519 68.7
TABLE-US-00031 TABLE 27 Absorbed Doses in mGy/MBq for the Human
Extrapolated from Mouse Model with isotope Lu-177, based on
[.sup.177Lu]Lu-BL02 in GRANTA519 xenograft mice. Organ Lu-177
Adrenals 0.00757 Brain 7.33e-05 Esophagus 0.00043 Eyes 0.000245
Gallbladder Wall 0.000863 Left colon 0.00141 Small Intestine
0.00131 Stomach Wall 0.000464 Right colon 0.00095 Rectum 0.000761
Heart 0.00148 Kidneys 0.0311 Liver 0.0185 Lungs 0.00333 Pancreas
0.000976 Prostate 0.000284 Salivary Glands 0.000254 Red Marrow
0.000343 Skeleton 0.000444 Spleen 0.00859 Testes 0.00245 Thymus
0.000336 Thyroid 0.00029 Urin Blad 0.000269 Remainder of the Body
0.000952
TABLE-US-00032 TABLE 28 Absorbed Doses in mGy/MBq for the Mouse 25
g model with isotope Lu-177, based on [.sup.177Lu]Lu-BL18 in Daudi
xenograft mice. Organ Lu-177 Brain 1.91 Large Intestine 3.41 Small
Intestine 3.42 Stomach 3.75 Heart 5.48 Kidneys 5.77 Liver 6.17
Lungs 7.63 Pancreas 4.2 Bone 1.27e+02 Spleen 11.3 Testes 8.79
Thyroid 2.55 Bladder 2.8 Remainder of the Body 2.88 Daudi
2044.55
TABLE-US-00033 TABLE 29 Absorbed Doses in mGy/MBq for the Human
Extrapolated from Mouse Model with isotope Lu-177, based on
[.sup.177Lu]Lu-BL18 in Daudi xenograft mice. Organ Lu-177 Adrenals
0.341 Brain 0.00596 Esophagus 0.025 Eyes 0.0198 Gallbladder Wall
0.0256 Left colon 0.0594 Small Intestine 0.0563 Stomach Wall 0.034
Right colon 0.0409 Rectum 0.0384 Heart 0.27 Kidneys 0.129 Liver
0.139 Lungs 0.229 Pancreas 0.0598 Prostate 0.0211 Salivary Glands
0.0204 Red Marrow 0.0733 Skeleton 0.044 Spleen 0.38 Testes 0.274
Thymus 0.0249 Thyroid 0.022 Urin Blad 0.021 Remainder of the Body
0.0341
TABLE-US-00034 TABLE 30 Absorbed Doses in mGy/MBq for the Mouse 25
g model with isotope Lu-177, based on [.sup.177Lu]Lu-BL19 in Daudi
xenograft mice. Organ Lu-177 Brain 1.8 Large Intestine 3.18 Small
Intestine 3.19 Stomach 3.39 Heart 5.29 Kidneys 5.43 Liver 5.71
Lungs 7.18 Pancreas 3.97 Bone 1.19e+02 Spleen 8.76 Testes 7.62
Thyroid 2.41 Bladder 2.6 Remainder of the Body 2.69 Daudi
1587.55
TABLE-US-00035 TABLE 31 Absorbed Doses in mGy/MBq for the Human
Extrapolated from Mouse Model with isotope Lu-177, based on
[.sup.177Lu]Lu-BL19 in Daudi xenograft mice. Organ Lu-177 Adrenals
0.245 Brain 0.00581 Esophagus 0.0242 Eyes 0.0193 Gallbladder Wall
0.0247 Left colon 0.056 Small Intestine 0.0532 Stomach Wall 0.0313
Right colon 0.0389 Rectum 0.0366 Heart 0.258 Kidneys 0.123 Liver
0.128 Lungs 0.215 Pancreas 0.0594 Prostate 0.0206 Salivary Glands
0.0199 Red Marrow 0.0688 Skeleton 0.0417 Spleen 0.279 Testes 0.232
Thymus 0.0241 Thyroid 0.0214 Urin Blad 0.0205 Remainder of the Body
0.0326
TABLE-US-00036 TABLE 32 Absorbed Doses in mGy/MBq for the Mouse 25
g model with isotope Lu-177, based on [.sup.177Lu]Lu-BL23 in Daudi
xenograft mice. Organ Lu-177 Brain 0.219 Large Intestine 0.54 Small
Intestine 0.627 Stomach 1.27 Heart 1.1 Kidneys 1.9 Liver 4.71 Lungs
1.58 Pancreas 1.13 Bone 8.97 Spleen 9.06 Testes 0.562 Thyroid 0.338
Bladder 0.289 Remainder of the Body 0.806 Daudi 709.31
TABLE-US-00037 TABLE 33 Absorbed Doses in mGy/MBq for the Human
Extrapolated from Mouse Model with isotope Lu-177, based on
[.sup.177Lu]Lu-BL23 in Daudi xenograft mice. Organ Lu-177 Adrenals
0.0379 Brain 0.000494 Esophagus 0.00347 Eyes 0.00176 Gallbladder
Wall 0.00665 Left colon 0.00724 Small Intestine 0.00665 Stomach
Wall 0.00491 Right colon 0.00536 Rectum 0.00406 Heart 0.0196
Kidneys 0.0501 Liver 0.152 Lungs 0.0365 Pancreas 0.00671 Prostate
0.00199 Salivary Glands 0.00184 Red Marrow 0.00529 Skeleton 0.00412
Spleen 0.363 Testes 0.0149 Thymus 0.00268 Thyroid 0.0022 Urin Blad
0.00191 Total Body 0.00753
TABLE-US-00038 TABLE 34 The in vivo stability of select peptides
Peptide Time Point % Intact Peptide n [.sup.68Ga]Ga-BL02 5 mins
>99% 3 [.sup.18F]F-BL08 5 mins >99% 3 [.sup.18F]F-BL09 5 mins
>99% 3 [.sup.177Lu]Lu-BL19 24 Hrs >98% 3 [.sup.177Lu]Lu-BL19
120 Hrs >93% 3
[0241] The present invention has been described with regard to one
or more embodiments. However, it will be apparent to persons
skilled in the art that a number of variations and modifications
can be made without departing from the scope of the invention as
defined herein.
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